PH-responsive artesunate polymer prodrugs with enhanced ablation effect on rodent xenograft colon cancer

Article abstract of DOI:10.2147/IJN.S242032

Purpose: In this study, pH-sensitive poly(2-ethyl-2-oxazoline)-poly(lactic acid)-poly(β-amino ester) (PEOz-PLA-PBAE) triblock copolymers were synthesized and were conjugated with an antimalaria drug artesunate (ART), for inhibition of a colon cancer xenog

Full text of DOI:10.2147/IJN.S242032

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Open Access Full Text Article
O R I G I N A L R E S E A R C H
pH-Responsive Artesunate Polymer Prodrugs
with Enhanced Ablation Effect on Rodent
Xenograft Colon Cancer
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Purpose: In this study, pH-sensitive poly(2-ethyl-2-oxazoline)-poly(lactic acid)-poly(β-
Dan-Li Hao
Ran Xie
Ge-Jing De
Hong Yi
Chen Zang
Mi-Yi Yang
Li Liu
Hai Ma
amino ester) (PEOz-PLA-PBAE) triblock copolymers were synthesized and were conjugated
with an antimalaria drug artesunate (ART), for inhibition of a colon cancer xenograft model.
Methods: The as-prepared polymer prodrugs are tended to self-assemble into polymeric
micelles in aqueous milieu, with PEOz segment as hydrophilic shell and PLA-PBAE
segment as hydrophobic core.
Results: The pH sensitivity of the as-prepared copolymers was confirmed by acid-base titration
with pKb values around 6.5. The drug-conjugated polymer micelles showed high stability for at
least 96 h in PBS and 37°C, respectively. The as-prepared copolymer prodrugs showed high drug
Wei-Yan Cai
loading content, with 9.57% 1.24% of drug loading for PEOz-PLA-PBAE-ART4. The con-
Qing-He Zhao
Feng Sui
Yan-Jun Chen
jugated ART could be released in a sustained and pH-dependent manner, with 92% of released
drug at pH 6.0 and 57% of drug released at pH 7.4, respectively. In addition, in vitro experiments
showed higher inhibitory effect of the prodrugs on rodent CT-26 cells than that of free ART.
Animal studies also demonstrated the enhanced inhibitory efficacy of PEOz-PLA-PBAE-ART2
micelles on the growth of rodent xenograft tumor.
Institute of Chinese Materia Medica,
China Academy of Chinese Medical
Sciences, Beijing 100700, People’s
Republic of China
Conclusion: The pH-responsive artesunate polymer prodrugs are promising candidates for
colon cancer adjuvant therapy.
Keywords: artesunate, micelles, pH responsive, polymer prodrug, poly(2-ethyl-
2-oxazoline), poly(β-amino ester)
Introduction
Colon cancer was regarded as one of the most common malignancies with high mobidity
and mortality worldwide, with estimated 1.1 million new cases and 551,269 deaths in
2018 worldwide,1 which posted great challenges to human life and health. Currently, the
mainstream clinical treatments for cancer are surgery, chemotherapy, and radiotherapy,
etc.2 Among which chemotherapy was considered as one of the main therapies for colon
cancer.3 However, the prognosis of chemotherapy is hardly optimistic due to the non-
targeted anti-cancer drug delivery to the tumor site and the severe toxic effects on normal
tissues, leading to limited therapeutic efficacy as well as drug resistance and/or migration
of the tumor cells to other organs, thus limiting the 5-year survival rates of patients.4
Therefore, it is urgent to develop novel drug delivery depots with tumor-specific drug
delivery characteristics and less toxic side effects on normal tissues.5
Correspondence: Qing-He Zhao;
Yan-Jun Chen
Tel/Fax +86-10-84036059
Email qhzhao@icmm.ac.cn;
yjchen@icmm.ac.cn
Artesunate (ART), a semi-synthetic derivative of artemisinin,6 has been recog-
nized as a safe and highly effective anti-malaria drug.7 In recent years numerous
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studies have revealed its anti-tumor effect on various can- Administration (FDA) of the USA with applications in the
cer cell lines,8 including multiple myeloma,9 chronic mye- fields of food, pharmaceuticals, etc.30 Biomaterials modified
loid leukemia,10 bladder cancer,11 breast cancer,12 head
with PEOz have shown enhanced circulation half-life,
and neck tumor,13 hepatocellular carcinoma,14 prostate
improved tumor accumulation, and reduced off-target
cancer,15 and colon cancer,16 etc. Several in vitro studies
effects. Poly-D,L-lactide (PLA) has been applied as
revealed that ART can induce the pleiotropic responses in
cancer cells, including inhibition of cell proliferation via
cell cycle arrest,17 apoptosis,18 induction of DNA
lesions,19 anti-angiogenesis,20 and modulation of nuclear
receptor responsiveness, etc. In addition, it has been
reported that artesunate exhibited promising advantages
for treatment of tumor, such as the selective inhibition
effect on multiple tumor cells, and less toxicity toward
healthy tissues.8 ART was also employed in combination
with other anticancer agents for synergistic therapy of
cancer.21 However, the applications of artesunate in clinic
are still limited due to its poor water solubility, short half-
life in blood circulation, low bioavailability, as well as
poor stability. Therefore, it is highly desirable to design
an ART depot with sustained release and targeted delivery
properties to further explore its applications in other indi-
cations such as tumor therapy.
a
hydrophobic segment in numerous drug delivery
systems.31 Poly(β-amino esters) (PBAE), synthesized by
Michael addition reaction using primary or secondary di-
amines and diacrylates, was extensively used for delivery
of drugs and siRNAs due to its pH-responsive properties.32
In this study, we prepared pH-sensitive poly(β-amino
ester) copolymers with subsequent conjugation with ART to
improve the therapeutic efficacy of ART against colon can-
cer. The drug cargo was constituted of PEOz as hydrophilic
corona and polylactide/PBAE as hydrophobic core
(Scheme 1). The copolymer exhibited proton sponge prop-
erties at pH below its pKb (~6.5) owing to the presence of
amine groups.33 The drug-loaded copolymer micelles could
be accumulated in tumor tissue owning to the EPR effect.34
In this work, the pH-responsive prodrug micelles showed
enhanced cellular internalization and stronger inhibitory
effect on CT-26 cells. Moreover, the ART-conjugated pro-
drugs were demonstrated with elevated therapeutic efficacy
on rodent xenograft tumors, thus highlighting their great
potential for applications in the treatment of colon cancer.
Nanoscale drug delivery systems (NDDS) using lipo-
somes, polymer micelles and nanoparticles, etc., have been
extensively investigated for targeted delivery of chemother-
apeutic drugs in the treatment of cancer due to their enhanced
permeability and retention (EPR) effect on tumor malignan-
cies. Bu reported pH and reduction dual-responsive micelles
to deliver doxorubicin (DOX), which showed excellent per-
formances in delivering DOX under pH and redox stimuli.22
NDDS can also offer improved pharmacokinetic properties,
controlled and sustained release of drugs, and more impor-
tantly, lower systemic toxicity. A variety of nanotechnology
has been developed in the past years to improve the solubility
and bioavailability of ART.23–26 ART loaded liposomes were
reported with glioma targeting capabilities with enhanced
toxicity towards glioma U87 cells.27 Jin et al reported arte-
sunate nanoliposomes, prepared with cholesterol and an
appropriate amount of lecithin via thin film hydration
method, showing stronger antitumor effect on HepG2 cells
than that of free artesunate.28
Materials and Methods
Materials and Cell Lines
N-Boc-ethanolamine was provided by Ark (Qingyuan,
Guangdong, China). Tin (II) 2-ethylhexanoate, and 4ʹ,6-dia-
midino-2-phenylindole (DAPI) were purchased from Sigma-
Aldrich (St. Louis, MO, USA). D,L-lactide was received from
Jinan Daigang (Jinan, Shandong, China). 2-Ethyl-2-oxazoline
(EOz), 4-toluene sulfonyl chloride, 2-amino-1,3-propanediol,
6-amino-1-hexanol, 1,6-hexanediol diacrylate and 4-dimethy-
laminopyridine were purchased from Aladdin (Shanghai,
China). Acryloyl chloride, 1,4-butanediol diacrylate, 1,9-bis-
(acryloyloxy) nonane and coumarin-6 (C6) were obtained
from TCI (Tokyo, Japan). 1-(3-(Dimethylamino) propyl)-
3-ethylcarbodiimide hydrochloride was provided by Adamas
(Shanghai, China). Artesunate (ART) was purchased
from MACKLIN (Shanghai, China). ART injection was
obtained from Guilin Nanyao (Guilin, Guangxi, China).
In recent years, stimuli-responsive nanoparticles, from
which the drug release can be triggered by pH, temperature
and light, etc., have been intensively investigated as smart
drug-delivery systems for various therapeutic applications.29
Poly(2-ethyl-2-oxazoline) (PEOz) is a hydrophilic polymer
with advantageous biocompatibility and water-soluble prop-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide (MTT) was purchased from Beyotime Biotechnology
(Shanghai, China). Fetal bovine serum (FBS), RPMI-1640
erties, which has been approved by the Food and Drug medium (RPMI) and trypsin-EDTA were obtained from
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Hao et al
ART
O
O
O
m
H
N
O
O
O
n
N
O
O
N
N
O
O
ART
O
x
O
O
O
ART
O
O
O
O
O
ART
Self assembly
Tumor tissue
Blood vessel
pH~6.5
Cancer cell
Scheme 1 The formation of polymeric micelles of the PEOz-PLA-PBAE polymer prodrugs and the cellular delivery of the ART-conjugated prodrugs.
Gibco Company (San Diego, CA, USA). All other chemicals purified through silica gel column with petroleum ether as
eluent and was precipitated in hexane and vacuum dried to
obtain white powder product (Yield 45.6%).
were of analytical or HPLC grade and were used as received.
Mouse CT-26 colon cancer cells (Shanghai Zhong Qiao Xin
Zhou Biotechnology Co, Ltd., Shanghai, China) were used
and incubated in RPMI-1640 medium supplemented with
10% FBS and 1% penicillin–streptomycin at 37°C, 5% CO2
and 95% humidity.
Synthesis of PEOz
PEOz was synthesized by ring-opening polymerization of
2-ethyl-2-oxazoline with Boc-OTs as initiator. Shortly, 1.2
g (3.34 mmol) of Boc-OTs and 30 g (0.30 mol) EOz were
dissolved in 100 mL acetonitrile, and the polymerization
was conducted at 100°C for 48 h under N2 protection. The
reaction was terminated by addition of 100 mL of 0.1
M KOH methanol solution. Then, the obtained polymer
was dialyzed (MWCO = 3500) against deionized water for
2 days, and was lyophilized (Yield 51.3%).
Polymer Synthesis and Characterization
Synthesis of Boc-OTs
Boc-OTs was employed as an initiator and was synthesized
by substitution reaction (Scheme 2). In brief, 1.0 g (6.2
mmol) of N-Boc-ethanolamine and 1.73 mL (12.4 mmol)
of triethylamine (TEA) were dissolved in 5 mL anhydrous
dichloromethane (DCM). After which 1.3 g (6.82 mmol) of
4-toluene sulfonyl chloride was dissolved in 5 mL DCM
and was dropwise added into the above solution under ice
bath. After 4 hrs of vigorous agitation, the ice bath was
removed, and the reaction was allowed to continue for an
Synthesis of PEOz-PLA
Poly (2-ethyl-2-oxazoline)-b-poly(lactide) (PEOz-PLA)
was synthesized by ring opening polymerization. Briefly,
1.0 g (6.94 mmol) of D, L-lactide and 1.67 g (0.42 mmol) of
PEOz were dissolved in 5 mL anhydrous toluene and were
additional 24 hrs. Then, the product was isolated and initiated by tin (II) octoate as catalyst at 130°C for 24 h. The
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CH
3 f
O
H
N
O
H
N
c
TEA, DCM
e
O
O
b
H3C
S
SO2Cl
d
OH
O
a
a
0¡æ, 4 h; RT, 24h
O
O
O
O
Boc-OTS
PTSC
N-BOC-ethanolamine
Boc-OTS
H
N
b
b
N
OH
Acetonitile
RT, 24h
KOH/MeOH
RT, 4h
N
n
Et
Et
d
O
EOz
c
O
PEOz
H
N
O
O
O
O
O
Sn(OCt)2 / C7H8
O
N
H
m
PEOz
e
n
O
130¡æ, 4 h; RT, 24h
O
O
O
PEOz-PLA
D,L-LA
O
H
Cl
TEA, DCM
O
g
N
O
N
PEOz-PLA
h
n
O
0¡æ, 4 h; RT, 24h
m
f
O
O
O
O
Acryloyl chloride
PEOz-PLA-A
OH
j
O
H2N
OH
H
N
O
O
O
O
N
HO
i
O
O
O
O
N
O
N
j
N
PEOz-PLA-A
m
HO
n
x
O
i
O
DMF, 50¡æ, 48h
O
OH
O
O
O
OH
PEOz-PLA-PBAE
ART
O
O
H
O
O
N
O
O
ART
n
O
PEOz-PLA-PBAE
N
m
N
DMAP, EDAC
O
O
O
x
O
O
ART
O
O
ART
O
O
O
H
O
ART
H
O
PBAE-ART
O
O
O
O
O
H2N
B1
B2
O
O
H
H
O
HO
OH
1,4-Butanediol diacrylate
A1
A2
k
H
O
O
H
O
O
l
2-Amino-1,3-propanediol
O
1,6-Hexanediol diacrylate
H2N
6-Amino-1-hexanol
OH
O
OH
O
O
B3
O
O
Artesunate (ART)
1,9-Bis(acryloyloxy)nonane
Scheme 2 The synthesis routes and composition of Boc-OTs, PEOz, PEOz-PLA, PEOz-PLA-PBAEs and PBAE-ART prodrugs.
copolymer PEOz-PLA was obtained by precipitation in evaporation. The terminal activated PEOz-PLA-A was
diethyl ether and vacuum drying. (Yield 85.4%)
purified by dissolution/precipitation for 3 times by DCM/
diethyl ether and vacuum dried. (Yield 89.6%)
Acetylation of PEOz-PLA
To activate the terminal hydroxyl groups of PEOz-PLA,
Synthesis of PEOz-PLA-PBAE Triblock Copolymers
0.58 g (0.1 mmol) of PEOz-PLA and 28 μL (0.2 mmol) of A 2.90 g (0.5mmol) of acrylic ester terminated PEOz-PLA
TEA were dissolved in 5 mL dichloromethane (DCM), and -A was dissolved in DCM and reacted with 5 mmol of
40.6 μL (0.5 mmol) of acryloyl chloride was added drop- 1,4-butanediol diacrylate (0.99 g) (or 1,6-hexanediol dia-
wise at 4°C and the reaction was allowed to continue crylate (1.13 g), or 1,9-bis(acryloyloxy) nonane) (1.35 g)
overnight. The solvent was removed via rotary and 5.5 mmol of 2-amino-1,3-propanediol (0.501 g) (or
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6-amino-1-hexanol (0.645 g)) at 50°C for 24 h. The final calculated against polymer concentration to determine the
product PEOz-PLA-PBAE was obtained by precipitation/ CMC of the respective copolymers.
dissolution in diethyl ether/DCM thrice and vacuum dry-
ing. (Yield 78.4%)
The size and zeta potential of the as-prepared micelles
were measured on a Malvern Zetasizer Nano ZS90
(Worcestershire, United Kingdom) with a detection angle at
90°. TEM images were obtained using a HITACHI H7650
Synthesis of ART-Conjugated Polymeric Prodrug
A 3.63 g (0.5 mmol) of PEOz-PLA-PBAE copolymer and (Tokyo, Japan) transmission electron microscope operated
0.96 g (2.5 mmol) of ART were dissolved in 15 mL DCM, with an acceleration voltage of 80 kV. The samples were
and 0.48 g (2.5 mmol) of 1-(3-(dimethylamino) propyl)- prepared by dipping a TEM grid into a 0.1 wt % sample
3-ethylcarbodiimide hydrochloride (EDAC) and 0.031 solution. The extra water was blotted with filter paper. The
g (0.25mmol) of 4-dimethylaminopyridine (DMAP) were drug grafting ratio (GR) was determined by cleavage of the
added into the solution to conjugate ART onto the side drug-conjugated polymers with esterase and was subjected to
chains of the copolymer. The solution was stirred at room high-performance liquid chromatography (HPLC, Agilent
temperature for 24 h. The solvent was removed by rotary 1260, Santa Clara, CA, USA) equipped with an Agilent C18
evaporation to terminate the reaction. The copolymer was column (4.6 × 250 mm, 5 mm, column temperature 30°C),
reconstituted by DMF and placed into dialysis tubing with a mobile phase of acetonitrile/water (45:55, v/v) and
(MWCO = 3500) and dialyzed against deionized water a flow rate of 1.0 mL/min. A fixed amount of lyophilized
for 2 days. The solution was filtered through 0.22 μm filter micelles was dissolved in methanol and subjected to HPLC
and vacuum dried to obtain ART-conjugated prodrugs.
and was detected via a UV detector at 216 nm. The content of
ART was determined by a calibration curve of ART in the
mobile phase. The grafting ratio (GR) was calculated accord-
Characterization of Copolymers and Prodrugs
1H NMR spectra of copolymers were obtained by a Bruker ing to the following equation:
AscendTM 600 MHz NMR spectrometer (Billerica, MA,
weight of ART in the prodrug
GR ¼
ꢀ 100%
USA) in CDCl3 at 25°C. The molecular weight of the
prodrugs was characterized using gel permeation chroma-
tography (GPC) equipped with a Waters Styrage™ HT3
GPC column (300 mm × 7.8 mm), a Waters 515 HPLC
pump and a Waters 2410 refractive index detector.
Tetrahydrofuran was used as mobile phase with a flow
rate of 1.0 mL/min. Narrow distributed polystyrene was
used as calibration standard. The pKb values of the as-
prepared copolymers were determined by acid/base titration
method. In a typical experiment, 20 mg of the polymer was
dissolved in 20 mL deionized water and the pH was
adjusted to less than 2.0, after that 20 μL of 0.1 N NaOH
was added and the solution pH was measured. The pKb was
plotted as the inflection point of the titration curve. The
critical micelle concentration (CMC) of the polymers was
determined by fluorescence measurements using pyrene
fluorescence spectroscopy according to our previous
weight of prodrug
In Vitro Drug Release
The release of ART from micelles was performed accord-
ing to a protocol reported previously.36 In short, 2 mL of
ART-conjugated micelles solution was sealed in a dialysis
bag (MWCO 3500) and was incubated in 15 mL of phos-
phate buffered saline (PBS) at pH 7.4 or pH 6.0. The drug
release was carried out at 37°C with an incubator shaking
horizontally at 100 rpm. The release medium was comple-
tely removed and supplemented with fresh PBS at prede-
termined time intervals. The ART concentration in the
medium was recorded with HPLC according to the afore-
mentioned protocol. The cumulative drug release was
plotted vs incubation time.
report.35 The emission wavelength was set at 390 nm with Cellular Uptake of Polymer Micelles
excitation slit and emission slit of 1.0 nm and 10.0 nm, Confocal laser scanning microscopy (CLSM) and flow
respectively. The excitation intensities at a wavelength cytometry were carried out to monitor the intracellular
range of 300–360 nm were monitored using a fluorescence uptake and distribution of drug-conjugated micelles. For
spectrophotometer (F-2000, HITACHI, Tokyo, Japan). The CLSM measurements, 1 × 105 CT-26 cells in 500 μL
concentration of the polymer solution was varied from RPMI 1640 were seeded onto glass coverslips which were
5.0×10−4 to 1.0 mg/mL containing 6×10−8 M of pyrene as placed in 6-well plates and incubated for 24 h. Then, the
a probe. The fluorescence intensity ratio of I336/I334 was cells were incubated for 0.5 h or 3 h with C6 or C6-loaded
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prodrug micelles (micelles concentration 50 μg/mL) which plates and incubated overnight. Then, the cells were treated
were dissolved in 500 μL RPMI 1640 supplemented with with free ART, PBAE-ART4, or PBAE-ART2, respectively,
10% FBS. At predetermined time points, the cells nuclei at ART dosage of 100 μM and then incubated for additional
were stained by 10 μg/mL DAPI (Invitrogen, Waltham, 24 h. The change of mitochondrial membrane potential of
MA, USA) for 15 min; then, the cells were washed with the cells was detected by incubation with a fluorescent
cold PBS and fixed with 4% paraformaldehyde. The cover- probe JC-1 at 37°C for 15 min. After washing with incuba-
slips were mounted onto glass slides and the cellular uptake tion buffer for three times, the cells were directly analyzed
was observed using confocal microscopy (Zeiss AXI0, by flow cytometry.
Oberkochen, Germany). For flow cytometry experiments,
the cells were first seeded and incubated with different C6
Cellular Reactive Oxygen Species (ROS)
Measurements
The released intracellular ROS in different groups was eval-
formulations in the same way as that in the CLSM observa-
tions. After incubation, the cells were washed with cold
PBS, trypsinized and resuspended in PBS. The suspended
uated using 6-carboxy-2ʹ, 7ʹ-dichlorodihydrofluorescein diace-
cells were directly introduced to a flow cytometer (BD
tate (DCFHDA, Qayee Bio, Shanghai, China). CT-26
FACS Calibur, San Jose, CA, USA) equipped with a 488
cells were seeded in 96-well plates with a density of 1 × 104
nm argon ion laser.
cells/well at 37°C and were incubated for 24 h. Then, the cells
were incubated with free ART, PBAE-ART4, or PBAE-ART2
In Vitro Cytotoxicity and Cell Apoptosis
with ART dosage of 100 μM for 24 h, respectively. The cells
Analysis
were collected and resuspended in 10 μM DCFHDA. After
To analyze the cytotoxicity of the as-prepared copolymer incubation at 37°C for 30 min, the fluorescence was measured
prodrugs, CT-26 cells were seeded in 96-well plates with using a microplate reader (excitation at 485 nm and emission
a density of 1 × 104 cells per well at 37°C under 5% CO2 at 530 nm).
and were subjected to incubation for 24 h. After that, the
cells were treated with fresh medium containing blank
Western Blot Analysis
polymer or polymer prodrugs with concentrations ranging
CT-26 cells were seeded in 6-well plates and were incubated
from 1 μM to 1000 μM. After 24 h, 48 h and 72 h of
with free ART, PBAE-ART4 or PBAE-ART2 (ART dosage
incubation, the cells viability was detected by a microplate
100 μM) for 24 h. Then, the cells were washed with cold PBS
reader (Thermo, MK3, Waltham, MA, USA) at an absor-
twice and incubated with lysis buffer containing 1 mmol
bance wavelength of 570 nm using MTT assay, respec-
phenylmethylsulfonyl fluoride for 30 min. A total of 30.0 μg
tively. The cellular viability data were analyzed using
of the proteins from different samples were added to the 10%
GraphPad Prism 5.01.
SDS–polyacrylamide gel to separate proteins with different
For cell apoptosis assay, cells were seeded in 12-well
molecular weights which were then transferred to the PVDF
plates at 2 × 105 cells/well. After 24 h of incubation,
membrane for 1 h, followed by blocking the membrane using
various ART formulations (ART dosage 100 μM) were
5% nonfat milk prepared with TBST (Tris buffered saline and
added to the cells medium and incubated for 24, 48 and
Tween 20) solution for 1 h at room temperature and incubation
72 h, respectively. Then, the cells were harvested, washed
with different primary antibodies of anti-cytochrome
twice with cold PBS and stained with Annexin V-FITC
c antibody (1:5000 diluted, 14 kDa, Abcam), anti-caspase-3
(BD Pharmingen, San Jose, CA, USA) and PI (BD
(1:1000 diluted, 46 kDa, Abcam) and anti-caspase-9 (1:1000
Pharmingen) for 15 min at room temperature under dark.
diluted, 32 kDa, Abcam) overnight at 4°C, respectively. Then,
Then, the treated cells were resuspended in PBS and were
the secondary antibodies anti-rabbit IgG (Abcam) and anti-
analyzed by flow cytometry (BD FACS Calibur).
mouse IgG (Abcam) were applied to incubate with the corre-
sponding antibodies for 45 min. After the membrane was
Mitochondrial Membrane Potential Assay washed with TBST for 10 min for three times, the bounded
Mitochondrial membrane potential was monitored using antibodies were detected with enhanced chemiluminescence
JC-1 Assay Kit (Beyotime Biotechnology) under flow cyto- reagents (Amersham, OH, USA) and the membrane was
metry following the manufacturer’s recommendations with exposed to hyperfilm (Amersham). The intensities of the
slight modifications. In brief, cells were seeded in 96-well proteins were measured by Image J (Tanon 5200) software
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Hao et al
and normalized to that of GAPDH and expressed as relative addition of diacrylate ester and amine groups (Scheme 2). The
ratios.
chemical structures of the copolymers were characterized by
1H NMR (CDCl3, 600 MHz) analysis, with corresponding
1
chemical shifts of the H NMR spectra as shown in (Figure
In vivo Tumor Inhibition Studies
S1). δ1.41 (a, Boc-OTs, -O(CH3)3), 3.38 (b, Boc-OTs, -OCH2
CH2-), 4.07 (c, Boc-OTs, -OCH2CH2-), 7.79 (d, Boc-OTs,
-C=CH-CH=C-), 7.35 (e, Boc-OTs, -C=CH-CH=C-), 2.45 (f,
Boc-OTs, =C-CH3) (Figure S1A); δ1.76 (a, PEOz, -C-
(CH3)3), 3.46 (b, PEOz, -N-CH2-CH2-), 2.40 (c, PEOz, -C-
CH2-CH3), 1.12 (d, PEOz, -C-CH2-CH3) (Figure S1B); δ5.20
(e, PLA, -CH-) (Figure S1C); δ5.91–6.19 (f, g, acryloyl,
=CH2) and δ6.47 (h, acryloyl, -O-CO-CH=) (Figure S1D);
δ4.16 (i, bisacrylate, -CH2-OC=O), 3.03 (j, N-methenyl,
N-CH-) (Figure S1E). It can be demonstrated that PEOz-
PLA-PBAE copolymers were successfully synthesized
according to the 1H NMR spectra.
Six-week female BALB/c mice (Academy of Military
Medical Sciences, Beijing, China) were used as animal
models. Animal studies were conducted according to the
regulation on experimental animals of China Academy of
Chinese Medical Sciences (CACMS), and were approved
by the animal care and use committee of the Institute of
Chinese Materia Medica, CACMS. CT-26 cells were
seeded subcutaneously (2×107 cells/mouse) into mice.
Mice were randomly divided into four groups (n = 5)
when the volume of tumors reached near 100 mm3. The
therapeutic effect of ART, PBAE-ART4 and PBAE-ART2
on the xenograft rodent tumor was analyzed by tail vein
injection of the respective ART formulations (ART dosage
of 20 mg/kg) every 2 days for 5 times, respectively. The
body weight and tumor volume were recorded and calcu-
lated every 2 days for 3 weeks. Mice were sacrificed at
21st day post-treatment and the primary tumors were
excised and weighted. The tumor volume (V) was calcu-
lated as follows: V = (L × W2)/2, where L and W were the
longest and the shortest diameter of the tumor.
The PBAE-ART prodrugs were synthesized through
esterification reaction between PELA-PBAE copolymers
and ART. The chemical structures of the copolymer pro-
1
drugs were characterized by H NMR analysis. The char-
acteristic chemical shift of ART could be easily identified
at δ 5.40 (l, artesunate, 1H) and 5.60 (k, artesunate, 1H)
(Figure S1F).
The PEOz-PLA-PBAE copolymers could be self-
assembled into micelles in aqueous solution. The critical
micelle concentration (CMC) of the copolymers was char-
acterized by pyrene fluorescence spectrometry to monitor
the self-assembly behavior of the copolymers. It should be
noted that the as-prepared copolymers exhibited very low
CMC values (Table 1, Figure S2), which is highly desir-
able when the formulations to be used for intravenous
administration. The sizes of the PELA-PBAE-ART
micelles increased significantly after conjugation with
ART, as compared to that of the PELA-PBAE copolymers,
indicating the more rigid molecular structure of the poly-
mer prodrugs due to introduction of the hydrophobic ART
molecules into the cores of the amphipathic copolymers
Statistical Analysis
Data were processed using GraphPad Prism 5.01 software
and presented as mean standard deviation (SD). Statistical
analysis was performed using one-way analysis of variance
(ANOVA). The difference was regarded statistically signif-
icant as *P < 0.05, very significant as **P < 0.01, and
extremely significant as ***P < 0.001.
Results
Synthesis and Characterization of
Polymers and Polymer Prodrugs
The preparation procedures used in the synthesis of polymer (Table 2). The morphology of ART-conjugated micelles
conjugates were depicted in (Scheme 2). The hydrophilic was characterized by TEM, which showed spherical
polymer PEOz was synthesized via ring opening polymeriza- morphologies of the micelles (Figure S3). It should be
tion (ROP) of 2-ethyl-2-oxazoline as initiated by noted that the size measured by TEM was smaller than
Boc-OTs. The polymerization process was quenched by that characterized by the dynamic light scattering (DLS),
methanolic potassium hydroxide to introduce hydroxyl group which should be ascribed to the shrinkage of the micelle
at the end of PEOz, which could be further conjugated with after drying,37 as compared to the stretching state of the
poly(D,L-lactide) (PLA) segment by ROP of D,L-lactide with micelle corona in the aqueous solution measured by DLS.
tin octoate as catalyst. Then, the copolymer was further end- Our results demonstrated that the formed polymeric
caped with poly(β-amino ester) (PBAE) segment by activation micelles have sizes smaller than 200 nm, which may
of PEOz-PLA with acryloyl chloride and following Michael facilitate the accumulation of the polymer prodrugs in
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tumor cells.38 To further evaluate the influence of pH on which was confirmed by the charge reversal of the
synthesized polymer prodrugs in acidic environments
(Figure S4A). From pH 6.0 to 5.0, the sizes of the micelles
decreased due to the electrostatic screening effect of the
excess protons in the surrounding milieu, leading to
decreased or diminished electrostatic repulsion between
the PBAE chains.39 As aforementioned, the ζ potentials
of the prodrugs increased significantly by decreasing the
surrounding pH from 7.4 to 6.5 (Figure S4B), due to the
protonation of the PBAE segment, and subsequent partial
charge neutralization.40,41
the size and surface charge of the polymeric micelles, the
as-prepared micelles were exposed to different incubation
milieu with pH at 5.0, 5.5, 6.0, 6.5 and 7.4, respectively,
and incubated at 37°C to monitor the changes of micelles’
size and zeta potential as measured by DLS (Figure S4). It
can be observed that the sizes of PELA-PBAE-ART1,
PELA-PBAE-ART2 or PELA-PBAE-ART3 micelles
increased slightly by decreasing the pH of the incubation
medium from 7.4 to 5.0. However, the sizes of PELA-
PBAE-ART4, PELA-PBAE-ART5, and PELA-PBAE-
ART6 micelles increased dramatically under similar
conditions. For example, the size of PELA-PBAE-ART4
micelles increased from 195 nm (pH 7.4) to 320 nm
(pH 6.0) and decreased dramatically from 320 nm (pH
6.0) to 205 nm (pH 5.0), which might be ascribed to the
protonation of PBAE segment in the pH range from 7.4 to
6.0, leading to the expansion of the micelles sizes due to
the electrostatic repulsion of the PBAE molecular chains,
The stability of the polymer prodrugs was investigated by
measurement of micelles’ sizes vs incubation time according
to our previous protocol.35 As shown in Figure S5, the sizes of
PELA-PBAE-ART2 prodrug showed high stability in the
investigated period, whereas the sizes of other prodrugs
increased to some extent during incubation. The drug grafting
ratios (GR) of the polymer prodrugs were ranged from 2.14
1.08% to 9.57 1.24%, respectively (Table 3), which was in
good agreement with the result calculated from the 1H NMR
data. The PEOz-PLA-PBAE copolymers were small sized and
narrowly distributed. However, significant increase in the
micelles’ sizes was observed after conjugation of the drug
onto the polymers backbone (Table 2). It should be noted
that the polymer prodrug (PBAE-ART1 and PBAE-ART4)
containing 1,4-butanediol diacrylate groups has higher drug
grafting ratio and larger micelle sizes. As a comparison, the
copolymer prodrug (PBAE-ART3 and PBAE-ART6) consist-
ing of 1,9-bis(acryloyloxy) nonane groups have lower drug
grafting ratio and smaller sizes. Whereas the copolymer
PBAE-ART2 consisting of 2-amino-1,3-propanediol and
1,6-hexanediol diacrylate showed high stability during incuba-
tion. Therefore, PBAE-ART4 and PBAE-ART2 prodrugs
were used in the subsequent in vitro and in vivo studies.
Table
1 Characterization of Poly(2-Ethyl-2-Oxazoline)-Poly
(Lactide)-Poly(β-Amino Ester) Copolymers (n=3)
Copolymers
Mn (1H- Mn
NMR)* (GPC)
PDI pKb CMC
(mg/L)
#
PEOz-PLA-PBAE-1 7249
PEOz-PLA-PBAE-2 8293
PEOz-PLA-PBAE-3 7153
PEOz-PLA-PBAE-4 7782
PEOz-PLA-PBAE-5 6985
PEOz-PLA-PBAE-6 6596
7113
7406
6626
6510
6570
6667
1.10 6.3
1.04 6.4
1.19 6.5
1.21 6.4
1.11 6.3
1.11 6.3
2.37
2.38
1.79
2.05
5.01
1.74
Notes: A1: 2-amino-1,3-propanediol, A2: 6-amino-1-hexanol, B1: 1.4-butanediol
diacrylate, B2: 1.6-hexanediol diacrylate, B3: 1.9-bis(acryloyloxy) nonane. PEOz-
PLA-PBAE-1 (A1B1), PEOz-PLA-PBAE-2 (A1B2), PEOz-PLA-PBAE-3 (A1B3) PEOz-
PLA-PBAE-4 (A2B1), PEOz-PLA-PBAE-5 (A2B2), PEOz-PLA-PBAE-6 (A2B3).
*Characterized by Mn (1H NMR, CDCl3 as solvent, 600 MHz). #GPC (eluent:
tetrahydrofuran, flow rate: 0.5 mL/min). The pKb values were determined by acid-
base titration. CMC was measured using pyrene fluorescence spectroscopy.
Abbreviations: CMC, critical micelle concentration; GPC, gel permeation chro-
matography; Mn, number average molecular weight; PDI, Polydispersity index; pKb,
Alkaline ionization equilibrium constant; PEOz, poly (2-ethyl-2-oxazoline); PLA, poly
(lactic acid); PBAE, poly(β-amino ester).
In Vitro Drug Release
The drug release behavior of ART-conjugated prodrugs was
investigated at 37°C in pH 7.4 or pH 6.0, respectively. As
Table 2 Variation of the Sizes of the PEOz-PLA-PBAE Copolymers Before and After Conjugation of ART (n=3)
Copolymers
Size (nm)
PDI
Polymer Prodrugs
Size (nm)
PDI
PEOz-PLA-PBAE-1
PEOz-PLA-PBAE-2
PEOz-PLA-PBAE-3
PEOz-PLA-PBAE-4
PEOz-PLA-PBAE-5
PEOz-PLA-PBAE-6
152.6 2.5
48.3 1.4
30.4 0.5
148.5 5.9
58.2 1.7
56.9 2.1
0.240
0.392
0.558
0.447
0.375
0.248
PBAE-ART1
PBAE-ART2
PBAE-ART3
PBAE-ART4
PBAE-ART5
PBAE-ART6
190.31 4.5
68.78 1.3
33.45 0.8
197.25 6.4
105.50 3.1
97.43 2.2
0.69
0.197
0.214
0.648
0.674
0.537
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Table 3 The Grafting Ratio (GR) of ART on the Respective
Polymers Prodrugs Determined by HPLC and 1H NMR,
Respectively (n=3)
stronger cytotoxicity against CT-26 cells, as a comparison
to that of free ART. As can be seen in Table 4, the IC50 of
PBAE-ART2 and PBAE-ART4 micelles were 1.94 fold and
1.37 fold lower than that of free ARTafter incubation for 72
h, respectively, indicating the stronger inhibitory efficacy of
the PBAE-ART2 micelles against CT-26 cells.
Polymer Prodrugs
GR (HPLC)
GR (1H-NMR)
PBAE-ART1
PBAE-ART2
PBAE-ART3
PBAE-ART4
PBAE-ART5
PBAE-ART6
5.31% 0.25%
4.96% 0.18%
3.98% 0.59%
9.57% 1.24%
3.79% 0.29%
2.14% 1.08%
6.43%
7.12%
5.74%
10.2%
5.52%
3.65%
Annexin V/PI staining was carried out to investigate
the influence of the apoptosis rates on CT-26 cells of
various ART formulations. The apoptosis rates for cells
treated with free ART, PBAE-ART4 and PBAE-ART2 for
24 h were 8.76%, 11.88% and 14.27%, respectively. After
incubation for 72 h, the apoptosis rates were increased to
18.41%, 22.49% and 26.85%, respectively, with stronger
apoptosis rates were observed in the micelles groups than
that of free drug (Figure 2). Moreover, an improved inhi-
bitory activity of PBAE-ART2 against that of PBAE-
ART4 was observed due to the enhanced cellular uptake
of the PBAE-ART2 prodrug (Figure 1A).
shown in Figure S6, similar release profiles of PBAE-ART4
and PBAE-ART2 micelles were observed at pH 7.4 and pH
6.0, respectively, with 57.2% and 55.9% of the drug released
from the PBAE-ART4 and PBAE-ART2 micelles after 48
h incubation at pH 7.4, respectively. However, 91.4% and
92.8% of ART was released from PBAE-ART4 and PBAE-
ART2 micelles at pH 6.0 within 48 h.
In Vitro Cellular Uptake
Mitochondrial Membrane Potentials and
Coumarin-6 (C6) was employed as a model drug to investi-
gate the cellular internalization of the as-prepared polymer
prodrugs. The uptake of PBAE-ART4-C6 or PBAE-
ART2-C6 micelles by CT-26 cells was observed by confocal
laser scanning microscopy (CLSM) and flow cytometry. As
shown in (Figure 1A), only weak fluorescence in the cyto-
plasm was observed after incubation with free C6 for 3 h. As
a comparison, stronger fluorescence in the cytoplasm was
illuminated in the cells incubated with PBAE-ART4-C6 or
PBAE-ART2-C6 micelles. The flow cytometry analysis also
demonstrated significant improvement of the fluorescence in
the micellar groups, as compared to that of the free fluores-
cent probe, demonstrating enhanced cellular uptake of
PBAE-ART4 and PBAE-ART2 micelles than that of free
C6 (Figure 1B). Moreover, the fluorescence of PBAE-
ART2-C6 micelles internalized by the cells was significantly
stronger than that of PBAE-ART4-C6 micelles, indicating the
improved internalization of smaller sized micelles by CT-26
cells.
Cellular ROS Analysis
CT-26 cells were harvested and stained with JC-1, and sub-
jected to flow cytometry analysis to determine the loss of
mitochondrial membrane potential (Δψm). The JC-1 probe
concentrates in the mitochondria with intact mitochondrial
membrane (high membrane potentials) as red fluorescent
aggregates, which will be released into cytoplasm in the
state of monomers with green fluorescence when the perme-
ability of mitochondria membrane increases (low membrane
potentials). Therefore, the red/green fluorescence ratio in the
cells was measured to demonstrate the loss of mitochondrial
membrane potential (Δψm) in this study. Our results demon-
strated that the Δψm of CT-26 cells decreased significantly
(1.85 fold and 14.75 fold) after incubation with PBAE-ART4
and PBAE-ART2 (Figure 3A) for 72 h, respectively, with
a comparison to free ART.
The formation of intracellular ROS in CT-26 colon
cancer cells was further measured after incubation with
different ART formulations (Figure 3B). It has been shown
that the intracellular ROS levels in the PBAE-ART4 and
PBAE-ART2 groups increased 1.43 fold and 1.83 fold,
respectively, as compared with free ART group.
Cytotoxicity Assay and Cellular
Apoptosis Analysis
The in vitro cytotoxicity of free ART, PBAE-ART4 and
PBAE-ART2 was evaluated by the MTT assay. Dose and
time-dependent manner was observed in the cytotoxicity Western Blot Analysis
analysis of free ARTand ART prodrugs (Figure S7), respec- The apoptosis of CT-26 cells was investigated by Western
tively, which was in accordance with previous studies.42 blotting analysis after incubation of the cells with various
The ART-conjugated prodrugs were demonstrated with ART formulations. It has been reported that cytochrome
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DAPI
Merge
A
C6
PBAE-ART4-C6
PBAE-ART2-C6
20 μm
B
150
100
50
C6
***
PBAE-ART4-C6
PBAE-ART2-C6
***
**
*
0
0
0.5
3
Time / h
Figure 1 Cellular uptake of free C6 and micelles incorporated C6. (A) Confocal FL images of cellular internalized C6, PBAE-ART4-C6 and PBAE-ART2-C6, after 3
h incubation with CT-26 cells. Blue FL represents DAPI, green FL represents C6. (B) Flow cytometry analysis of mean FL intensity (n=10,000 cells) in CT-26 cells incubated
with C6, PBAE-ART4-C6 and PBAE-ART2-C6 for 0.5 h and 3 h, respectively. Data are presented as the mean of three measurements SD. *P<0.05, **P<0.01, ***P<0.001.
c may upregulate the expression of caspase 9 as well as caspase administration. As shown in Figure 5A, significant differ-
3 in the mitochondrial apoptosis pathway.43 It was illustrated
ence in the tumor volume of various therapeutic groups can
that the expression of apoptosis-associated proteins such as
cytochrome c, caspase 9 and caspase 3 increased significantly
after CT-26 cells were treated by PBAE-ART4 and PBAE-
ART2, respectively, as compared to the free drug (Figure 4).
be observed. The tumor volumes were 542.5, 690.5 and
727.8 mm3 in PBAE-ART2, PBAE-ART4 and ART group
in the 21st day post-treatment, respectively, as compared to
834.9 mm3 of the saline group, indicating enhanced inhibi-
tory effect of the as-prepared prodrugs. No significant dif-
ference in the body weight of mice was observed in the
various ART formulations, demonstrating its high biocom-
patibility of the polymer prodrugs (Figure 5B). On the other
In vivo Tumor Inhibition Studies
In the current study, rodent CT-26 xenograft tumor model
was established to evaluate the anticancer efficacy of various
ART formulations on BALB/c mice via intravenous hand, PBAE-ART2 showed better efficacy than that of
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Table 4 The Half Inhibition Concentration (IC50) of Different ART
Formulations on CT-26 Cells After Incubation for 24, 48 and 72 h,
Respectively
chromatography (GPC). It can be seen that the as-prepared
copolymers were narrowly distributed with Mn (GPC)
slightly less than Mn (NMR), which may be ascribed to
the different chemical structure of polystyrene (PS) used
for calibration of the molecular weight of the triblock
copolymers by GPC (Table 1).
Drug Formulation
IC50(µM)
24h
48h
72h
ART
247.9 18.86
157.7 23.63
91.76 14.92
219.3 29.45
129.9 18.88
83.94 1.806
103.2 4.18
75.31 4.07
53.31 8.74
The synthesized PEOz-PLA-PBAE copolymers exhib-
ited a sharp pH change in the pH range from 4 to 10
(Figure S8), suggesting the pH-sensitive characteristics of
the copolymers. The pKb values of the copolymers with
different structures ranged from 6.3 to 6.5, as shown in
Table 1 and Figure S8. It has been reported that most solid
tumors exhibited weak acid environment,44 with even
lower pH values observed in the organelles of tumor
cells.45 The pH-triggered release of the drug from the
nano-drug carriers plays an important role in their applica-
tions in tumor therapy.46 It would be highly desirable to
release the conjugated drug rapidly in the mild acidic
tumor environment.47 The current study demonstrated the
accelerated drug release of the polymer prodrugs under
mild acidic conditions, which could be ascribed to hydro-
phobic/hydrophilic transition of the PBAE block at lower
pH values due to the protonation of the amino groups,47
resulting in the stretching state of ART linked PBAE
chains and accelerated release of the conjugated drug.48
It should be noted that no significant difference was
PBAE-ART4
PBAE-ART2
PBAE-ART4 (Figure 5C), hinting the pH-sensitive PBAE-
ART2 prodrug may promote the accumulation of the anti-
cancer drug in tumor cells, which is consistent with the
confocal microscopy experiments (Figure 1A).
Immunohistochemical Analysis
The therapeutic efficacy of the ART prodrugs was confirmed
again by histological analysis. As shown in Figure 6, tissue
necrosis and vacuolization could be observed in tumor tis-
sues after treatment with various ART formulations, as
compared with control group. Moreover, the ART prodrugs
showed better inhibitory efficacy on the xenograft tumor,
which may be ascribed to the enhanced penetration of the
polymer conjugated prodrugs into tumors.
Discussion
The number average molecular weight (Mn) of the copo-
1
lymers was characterized by H NMR and gel permeation observed in the drug release profiles of PBAE-ART4 and
A
B
24h
48h
72h
40
30
20
10
0
control
ART
Control
***
*
PBAE-ART4
PBAE-ART2
***
*
ART
***
**
PBAE-ART4
PBAE-ART2
24
48
72
Time / h
Figure 2 The apoptosis rates of CT-26 cells after incubation with various ART formulations. (A) Flow cytometry analysis via Annexin V/PI staining, (B) Quantitative analysis
of tumor cells apoptosis after incubation with ART formulations for 24, 48 and 72 h, respectively. *P<0.05, **P<0.01, ***P<0.001.
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A
B
70
***
60
50
40
30
20
10
0
250
200
150
100
50
control
ART
PBAE-ART4
PBAE-ART2
**
*
*
**
*
*
0
control
ART
PBAE-ART4 PBAE-ART2
24
48
72
Time / h
Figure 3 (A) The decrease of mitochondrial membrane potential after incubation of CT-26 cells with ART formulations for 24, 48 and 72 h, respectively. (B) The increased
ROS level in CT-26 cells after challenge with various ART drugs. *P<0.05, **P<0.01, ***P<0.001.
A
B
PBS
ART PBAE-ART4 PBAE-ART2
control
5
4
3
2
1
0
***
***
ART
***
PBAE-ART4
PBAE-ART2
Cytochrome c
***
***
**
Caspase9
Caspase3
Cytochrome c
Caspase9
Caspase3
GAPDH
Figure 4 Western blotting analyses of CT-26 cells after incubation with various ART formulations for 24 h. (A) The expression of cytochrome c, caspase 9 and caspase 3 in
CT-26 cells, GAPDH as control, (B) Relative intensities of the expression of the proteins in the cells. **P<0.01, ***P<0.001.
PBAE-ART2 micelles, which might be ascribed to the compared to that of free ART (Figure 3A). It can be hypothe-
similar chemical structure of the two prodrugs.
sized that the ART prodrug may trigger mitochondrial apop-
totic pathway in CT-26 cells. Moreover, a significant
decrease of ROS level was observed in the cells treated
with ART prodrugs (Figure 3B). It has been shown that
intracellular ROS plays an important role in cell death by
inducing apoptosis.52 The excessive ROS may induce oxida-
The mitochondria play a critical role in cell metabolism,
homeostasis and apoptosis, as well as cellular autophagy.49,50
The enhanced permeability of mitochondrial membrane (or
loss of mitochondrial membrane potential) is a marker event
in the process of cellular apoptosis.51 In this study, the
mitochondrial membrane potential of CT-26 cells decreased tive stress on mitochondria and destruction of the integrity of
significantly after incubation with ART prodrugs, as the mitochondria membrane structure, and induce
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A
B
1000
24
22
20
18
16
Control
Control
ART
ART
PBAE-ART4
PBAE-ART2
PBAE-ART4
800
600
400
200
0
PBAE-ART2
1
3
5
7
9
11 13 15 17 19 21
1
3
5
7
9
11 13 15 17 19 21
Time / Days
Time / Days
250
C
***
200
150
100
50
**
0
Control
ART
PBAE-ART4 PBAE-ART2
Figure 5 In vivo tumor inhibitory effects of various ART formulations on CT-26 xenograft tumors. (A) The tumor volume of CT-26 tumors post-treatment by ART
formulations via i.v. administration, (B) The body weight of mice in various groups post-treatments, (C) Weight of tumors of each therapeutic group on the 21st day post-
therapy. **P<0.01, ***P<0.001.
accelerated release of cytochrome c for activation of cellular artemisinin dimer in lipid nanoparticles showing enhanced
apoptosis.53 It has been indicated that the ART-conjugated anti-tumor efficacy against xenograft tumor.55 In this
prodrugs were more effective in induction of ROS than that study, the in vivo anti-tumoral effect of the ART prodrugs
of free ART, which may be ascribed to the elongated half-life was analyzed on CT-26 xenograft tumor-bearing mice. As
(data not shown) and the sustained release manner of the can be seen in Figure 5, the pH-responsive polymer pro-
prodrugs.6
drugs showed significant inhibitory efficacy on the growth
It was demonstrated that the ART-conjugated prodrugs of tumors through the enhanced cellular uptake of the drug
showed higher potency in upregulation of apoptosis- depot by the cancer cells (Figure 1), which was consistent
associated proteins and promotion of the apoptosis of with our previous studies.35 The histopathological analysis
CT-26 cells (Figure 4). It was revealed that the released also confirmed the elevated therapeutic effect of the poly-
cytochrome c can bind to Apaf, and the formed cyto- meric micelles by promoting apoptosis of CT-26 cells.
chrome c/Apaf complex can activate the expression of
caspase-9, which activates the downstream caspase-3 for Conclusion
induction of apoptosis in CT-26 cell lines.54
In this study, artesunate was conjugated with pH-sensitive
It has been reported that nano-sized drug delivery poly(2-ethyl-2-oxazoline)-poly(lactic acid)-poly(β-amino
systems are advantageous in tumor therapy with prolonged ester) triblock copolymers for accumulated delivery of
drug circulation time and enhanced accumulation of drug the drug to tumor cells and pH stimulated drug release
in tumor tissues.46 Zhang et al reported a pH-responsive under tumor acidic microenvironment. The enhanced
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PBS
ART
50 μm
50 μm
PBAE-ART4
PBAE-ART2
50 μm
50 μm
Figure 6 Histopathology analyses of tumor tissues of BALB/c mice in different ART formulation groups.
internalization of the pH-responsive polymer micelles was
Disclosure
confirmed by CLSM. The ART prodrugs showed better
efficacy in induction of apoptosis of CT-26 cells, as
reflected by flow cytometry analysis. The prodrug PBAE-
ART2 showed better antitumor effect through the induc-
tion of ROS production, and the enhanced expression of
tumor apoptosis-associated proteins, as compared with the
free drug. The efficacy of ART prodrugs was also con-
firmed at animal level with a rodent colon cancer model.
Results showed the as-prepared ART prodrugs exhibited
high potential for colon cancer adjuvant therapy.
The authors report no conflicts of interest in this work.
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