Self-assembly of oxidation-responsive polyethylene glycol-paclitaxel prodrug for cancer chemotherapy

Article abstract of DOI:10.1016/j.jconrel.2020.02.038

Amphiphilic drug conjugates can self-assemble into nanovehicles for cancer drug delivery, but the key is to design stable yet intracellular labile drug linkers for drug retention during blood circulation but fast intracellular drug release. The conjugatio

Full text of DOI:10.1016/j.jconrel.2020.02.038

Journal Pre-proof
Self-assembly of oxidation-responsive polyethylene glycol-
paclitaxel prodrug for cancer chemotherapy
Chengyuan Dong, Quan Zhou, Jiajia Xiang, Fusheng Liu,
Zhuxian Zhou, Youqing Shen
PII:
S0168-3659(20)30128-0
DOI:
https://doi.org/10.1016/j.jconrel.2020.02.038
COREL 10191
Reference:
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
17 December 2019
19 February 2020
24 February 2020
Please cite this article as: C. Dong, Q. Zhou, J. Xiang, et al., Self-assembly of oxidation-
responsive polyethylene glycol-paclitaxel prodrug for cancer chemotherapy, Journal of
Controlled Release (2020), https://doi.org/10.1016/j.jconrel.2020.02.038
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
©
2020 Published by Elsevier.

Journal Pre-proof
Self-Assembly of Oxidation-Responsive Polyethylene
Glycol-Paclitaxel Prodrug for Cancer Chemotherapy
1
ǂ
2ǂ
2
1
2
Chengyuan Dong , Quan Zhou , Jiajia Xiang *, Fusheng Liu *, Zhuxian Zhou *, and
2
Youqing Shen
1
. Brain Tumor Research Center, Beijing Neurosurgical Institute, Capital Medical
University; Department of Neurosurgery, Beijing Tiantan Hospital Affiliated to
Capital Medical University; Beijing Laboratory of Biomedical Materials, Beijing
1
2
00070, P. R. China
. Center for Bionanoengineering and Key Laboratory of Biomass Chemical
Engineering of Ministry of Education, College of Chemical and Biological
Engineering, Zhejiang University, Hangzhou, China 310007
*
Corresponding
author’s
E-mail
address:
xiang_jj@zju.edu.cn
or
liufushengs@hotmail.com, or zhouzx@zju.edu.cn
ǂ Equal contributors
Abstract:
Amphiphilic drug conjugates can self-assemble into nanovehicles for cancer drug
delivery, but the key is to design stable yet intracellular labile drug linkers for drug
retention during blood circulation but fast intracellular drug release. The conjugation
of paclitaxel (PTX) is generally via the ester of its 2'-hydroxyl group, but the ester is
either too stable to release PTX in the cytosol or so labile that hydrolyzes during
circulation. Herein, we report a p-(boronic ester)benzyl-based tumor-specifically
cleavable linker for preparing PTX-conjugate with polyethylene glycol (PEG, Mw =
5
000 Da) (PEG-B-PTX). The amphiphilic PEG-B-PTX self-assembled into micelle
with an average size of ~50 nm and a PTX loading content of 13.3 wt%. The
PEG-B-PTX micelles were very stable at the normal physiological environment and
thus circulated long in the blood compartment, but fast dissociated and released PTX
in response to the elevated reactive-oxygen species (ROS) level in tumors. The

Journal Pre-proof
conjugate micelles showed significantly improved antitumor efficiency in vitro and in
vivo against human glioma and breast cancer cells, and reduced toxicity compared to
the clinically used Taxol. Thus, the PTX-conjugate micelles were characteristic of
well-characterized chemical structure and nanostructure, precise and reproducible
drug loading efficiency (i.e., 100%) and fixed loading content, high PTX loading
content due to PTX itself as part of the carrier, no burst drug release, and easy and
reproducible fabrication of the micelles, which are all essential for clinical translation.
Keywords: ROS-responsive linker, polymer-drug conjugate, paclitaxel prodrug,
self-assembly prodrug, prodrug micelles.
1
. Introduction
Paclitaxel (PTX) is a potent antineoplastic agent for the treatment of various
cancers, including breast, lung, and ovarian carcinoma [1, 2]. However, its low water
solubility, significant systemic toxicity, and acquired drug resistance are its drawbacks
in clinical applications. Various PTX nanoformulations have been developed to
address these challenges [3-7]. For instance, Abraxane—albumin-bound PTX
nanoparticles—shows better pharmacokinetics and enhanced antitumor activity
compared to Taxol and was approved by the FDA for the treatment of metastatic
breast cancer in 2005 [8]. Another formulation, Genexol-PM, which is PTX loaded in
polyethylene glycol-block-polylactide (PEG-PLA) block copolymer micelles, has
been marketed in Asia and is in phase III clinical trial in the USA for the treatment of
metastatic breast cancer [9, 10]. Nevertheless, current clinically used PTX
nanoformulations generally have drawbacks of poor in vivo stability, premature
release, and only achieve minor improvements in efficacy [11, 12]. Therefore, great
efforts have been devoted to designing novel PTX delivery systems responsive to the
biological stimuli to control the drug retention/release for improved efficacy and
reduced side effects [13-18]; for instance, PTX was conjugated to polymers via labile
linkers in response to the tumor microenvironment stimuli such as such as pH [7, 19,
2
0], reactive oxygen species (ROS) [21-23], glutathione (GSH) [4, 24, 25], and
enzymes [26]. Several PTX prodrug formulations now have progressed to clinical

Journal Pre-proof
trials [3].
We demonstrated that a hydrophobic drug conjugated with a short water-soluble
polymer or peptide chain made the drug amphiphilic and thus self-assembled into
nanovehicles for delivery of the drug and even codelivery of other drugs [14, 16, 27].
This amphiphilic prodrug approach shows many advantages, including
well-characterized chemical structures, precise and reproducible drug loading
efficiency (i.e. 100%), fixed and high drug loading contents due to the drugs
themselves as part of the carriers, and easy and reproducible fabrication of the
nanovehicles. Very importantly, these nanovehicles are free of burst release problem
generally found in physically drug-loaded micelles [27]. Such characters are favorable
for clinical translation compared to complicated polymer counterparts [14, 16, 27-29].
Therefore, we proposed that making PTX amphiphilic with short polyethylene glycol
(
sPEG) would self-assemble into such micelles with the aforementioned characters
ready for clinical translation. The conjugation site for PTX is generally via its ester of
'-OH [30]. In our preliminary work, we prepared sPEG-PTX prodrugs via the 2'-OH
2
esters of different carboxylic acids. The prodrugs using the aliphatic acid esters, e.g.,
succinyl ester, as the linker could form well-defined micelles, but they had a very
short blood circulation times due to the fast hydrolysis of these esters, while the
prodrugs using the esters of aromatic acids were too stable to hydrolyze even in the
presence of esterase. Therefore, a stable but intracellular labile ester of PTX is the key
for the PTX prodrug forming stable and long-circulating nanovehicles to maximize
therapeutic efficacy and to minimize side effects.
Herein, we report a PEG-B-PTX prodrug with a benzylboronic ester-based
ROS-responsive cleavable linker between PEG and PTX (Scheme 1). Very
interestingly, the p-(boronic ester)benzyl succinyl ester of PTX was very stable, and
the PEG-B-PTX was hardly hydrolyzable in normal physiological conditions. On the
other hand, benzylboronic ester is known oxidizable by the elevated levels of ROS in
tumor and tumor cells [31, 32] to trigger an elimination reaction [33-35]. Thus, the
amphiphilic PEG-B-PTX could self-assemble into stable micelles with a size of about
5
0 nm and circulated long in blood. Once accumulated in tumor, the high

Journal Pre-proof
concentration of ROS oxidized the p-(boronic ester)benzyl group and induced the
subsequent 1,4-elimination reaction, resulting in self-cleavage of the linker to release
PTX and quinone as a by-product. The released quinone could deplete intracellular
GSH, synergistically killing cancer cells [36, 37]. Compared with commercial Taxol
and non-responsive control prodrug, PEG-B-PTX exhibited prolonged circulation
time, improved in vivo antitumor efficacy, and low side effects on mouse tumor
xenograft model. With the aforementioned characters, this conjugate has high
potentials for clinical translation.
Scheme 1. The chemical structure of the PTX prodrug, PEG-B-PTX, and it's
self-assembly into prodrug micelles for the tumor ROS-triggered drug release.
2
2
. Materials and methods
.1. Materials

Journal Pre-proof
2
-Amino-4'-bromoacetophenone hydrochloride, di-tert butyl dicarbonate, sodium
borohydride (NaBH4), bis(pinacolato)diboron, potassium acetate (KOAc),
.
1
'1-bis(diphenylphosphino) ferrocene palladium(II) chloride (Pd(dppf)Cl2 DCM),
succinic anhydride, trifluoroacetic acid (TFA), 4-pentynoic acid (PA),
N-hydroxysuccinimide
-(dimethylamino)pyridine
(NHS),
N,N-dicyclohexylcarbodiimide
(DMAP), CuBr,
(DCC),
and
4
N,N,N,N,-pentamethyldiethylenetriamine (PMDETA) were purchased from Energy
Chemical (Shanghai). Paclitaxel (PTX) was purchased from Huilin Bio-tech Co., Ltd.
(
Xi’an, China). Methoxypoly(ethylene glycol) with a molecular weight of ~5,000
mPEG5K-OH) was purchased from Sigma-Aldrich (Shanghai, China). All other
organic solvents were purchased from Sinopharm Chemical Reagent Co. Ltd.
Shanghai, China). Dichloromethane (CH2Cl2), tetrahydrofuran (THF), N,
(
(
N-dimethylformamide (DMF), dioxane, and triethylamine (TEA) were dried over
calcium hydride (CaH2) and distilled before use.
Fetal bovine serum (FBS), trypsin, and RPMI 1640 medium were purchased
from GIBCO. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and ROS assay kit (2′,7′-dichlorofluorescein diacetate, DCFH-DA) were purchased
from Beyotime Institute of Biotechnology (Shanghai, China). U251 cells were
purchased from the Cell Bank of Peking Union Medical College (Beijing, China).
Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) with 10 % fetal
bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL
streptomycin at 37°C in an environment of 5% CO2. Human breast cancer cell line
MCF-7 cells were purchased from American Type Culture Collection (Manassas,
USA). Cells were incubated in RPMI-1640 medium containing 10% heat-inactivated
FBS and 1% penicillin/streptomycin solution in a humidified atmosphere of 5% CO2
at 37 ºC. Female BALB/c mice (6-8 weeks) were purchased from the Animal Center
of Zhejiang University and maintained under standard conditions. Animal
experiments were approved by the Animal Care and Use Committee of Zhejiang
University and were carried out in accordance with the institutional guidelines.

Journal Pre-proof
2
.2. Synthesis of PEG-B-PTX (Scheme 2)
2
.2.1 Synthesis of compound 2
Compound 1 (5 g, 23.3 mmol) and di-tert-butyl dicarbonate (5.6 g, 25.7 mmol)
were dissolved in 100 mL THF at 0 ºC followed by the addition of Et3N (7.5 mL, 54.1
mmol) in 50 mL THF with a dropping funnel. After the addition was completed, the
reaction mixture was heated to 40ºC and stirred for another 2 h. After removing the
salts by filtration, the solvent was evaporated, and another 150 mL ethyl acetate was
added to dissolve the residues. The organic solution was washed with saturated NH4Cl
aqueous solution, NaHCO3 aqueous solution, and brine, and then dried over
anhydrous Na2SO4. After removing the solvent, the residues were further purified by
1
flash chromatography affording compound 2 as a white solid (11.5 g, yield: 90%). H
NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 5.50 (s,
1
H), 4.61 (d, J = 4.4 Hz, 2H), 1.47 (s, 9H).
Similarly, compound 8 was obtained as a white solid (yield: 73.4%). H NMR
400 MHz, CDCl3) δ 8.00 – 7.88 (m, 2H), 7.65 – 7.56 (m, 1H), 7.48 (dd, J = 10.6, 4.7
Hz, 2H), 5.56 (s, 1H), 4.66 (d, J = 4.4 Hz, 2H), 1.47 (s, 9H).
1
(
2
.2.2 Synthesis of compound 3
To a solution of compound 2 (11.5 g, 36.6 mmol) in methanol, NaBH4 (7.0 g,
85 mmol) was added portion-wise over 10 minutes with vigorous stirring in an
1
ice-salt bath. The mixture was stirred until the TLC monitoring indicated that the
reaction of compound 2 was complete. The reaction was next quenched with water
addition. Methanol was removed by evaporation, and the aqueous solution was
extracted with ethyl acetate (50 mL × 3). The combined organic phase was washed
with brine, dried over anhydrous Na2SO4, and filtered. After concentration, the crude
product was recrystallized in hexane to give compound 3 as a white crystal (10.7 g,
1
yield: 92%). H NMR (400 MHz, CDCl3) δ 7.54 – 7.40 (m, 2H), 7.24 (d, J = 8.4 Hz,
2
1
H), 4.93 (s, 1H), 4.79 (dd, J = 7.2, 2.5 Hz, 1H), 3.51 – 3.34 (m, 1H), 3.31 – 3.09 (m,
H), 1.44 (s, 9H).
1
Compound 9 was similarly obtained as a white crystal (yield: 89.2%). H NMR
(400 MHz, CDCl3) δ 7.41 – 7.26 (m, 5H), 4.96 (s, 1H), 4.88 – 4.74 (m, 1H), 3.46 (s,

Journal Pre-proof
1
2
H), 3.32 – 3.15 (m, 1H), 1.44 (s, 9H).
.2.3 Synthesis of compound 4
Compound 3 (7.6 g, 24.0 mmol), bis(pinacolato)diboron (21.4 g, 84.3 mmol),
.
Pd(dppf)Cl2 DCM (1.6 g 2.19 mmol), and potassium acetate (12.4 g, 126 mmol) were
dissolved in 100 mL 1,4-dioxane under an argon atmosphere. The reaction was heated
to 90 ºC and stirred for 12 h. After cooling to room temperature, the undissolved
potassium acetate was removed by filtration. After removal of 1,4-dioxane under
reduced pressure, the reaction mixture was diluted with EtOAc (150 mL) and washed
several times with 1 N HCl and brine. The organic phase was dried over Na2SO4,
filtered, and the solvent was removed by evaporation. The crude product was purified
1
with flash chromatography to give compound 4 as a white solid (6.5 g, 74.7%). H
NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 4.96 –
4
2
.77 (m, 2H), 3.49 (s, 1H), 3.30 – 3.07 (m, 2H), 1.44 (s, 9H), 1.34 (s, 12H).
.2.4 Synthesis of compound 6
4
-Pentynoic acid (5.00 g, 51.0 mmol), NHS (6.40 g, 55.6 mmol), and DMAP
(1.24 g, 10.1 mmol) were dissolved in 100 mL CH2Cl2 at 0 °C followed by the
addition of DCC (12.6 g, 61.2 mmol) in 50 mL CH2Cl2. The reaction mixture was
then warmed to room temperature and stirred for another 2 h. After filtration, the
solution was washed with 1 N HCl, saturated NaHCO3 aqueous solution, and brine.
The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The
crude product was recrystallized in ethyl acetate/hexane to give PA-NHS as a white
crystal with a yield of 76.4%.
Compound 4 (0.50 g, 1.38 mmol) was dissolved in a mixture of CH2Cl2/TFA (6
mL, 2:1) and stirred for 6 h at room temperature. Once TLC monitoring indicated
complete deprotection, the solvent was removed and additional dry CH2Cl2 was added
with 1 mL Et3N. A solution of 5 mL CH2Cl2 containing NHS-activated 4-pentynoic
acid (PA-NHS, 0.26 g, 1.33 mmol) was added dropwise. After stirring at room
temperature for 1 h, the mixture was diluted with 50 mL CH2Cl2 and washed
successively with 1 N HCl, saturated NaHCO3 aqueous solution, and brine. The
separated organic phase was dried over Na2SO4, filtered, and concentrated. The

Journal Pre-proof
resulting residue was further purified by flash chromatograph to give compound 6 as a
1
white solid (0.33 g, yield: 72.2%). H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.0 Hz,
2
H), 7.36 (d, J = 7.9 Hz, 2H), 6.14 (s, 1H), 4.85 (dd, J = 7.6, 3.3 Hz, 1H), 3.71 (ddd, J
14.1, 6.8, 3.4 Hz, 1H), 3.31 (ddd, J = 13.8, 7.6, 5.0 Hz, 1H), 2.72 (s, 1H), 2.56 –
=
1
3
2
.46 (m, 2H), 2.38 (t, J = 7.1 Hz, 2H), 1.99 (t, J = 2.6 Hz, 1H), 1.33 (s, 12H). C
NMR (101 MHz, CDCl3) δ 172.41 (s), 144.73 (s), 135.03 (s), 125.16 (s), 83.88 (s),
2.81 (s), 73.51 (s), 69.53 (s), 47.38 (s), 35.17 (s), 24.87 (s), 14.86 (s).
Compound 11 was similarly obtained as a white solid (yield: 68.0%). H NMR
8
1
(400 MHz, CDCl3) δ 7.42 – 7.28 (m, 5H), 6.76 (s, 1H), 5.83 (s, 1H), 4.83 (s, 1H), 3.62
1
3
–
1
3
2
3.28 (m, 4H), 1.40 (d, J = 14.3 Hz, 9H). C NMR (101 MHz, CDCl3) δ 172.34 (s),
41.63 (s), 128.57 (s), 127.93 (s), 125.86 (s), 82.84 (s), 73.47 (s), 69.51 (s), 47.42 (s),
5.19 (s), 14.87 (s).
.3.5 Synthesis of PA-B-PX
PTX and succinic anhydride were first reacted in anhydrous pyridine to afford
PTX-SA. PTX-SA (0.92 g, 0.96 mmol), compound 6 (0.33 g, 0.96 mmol), and DMAP
20 mg, 0.16 mmol) were dissolved in 30 mL dry CH2Cl2 at 0 °C followed by the
(
addition of DCC (0.30 mg, 1.46 mmol) in 10 mL CH2Cl2. The reaction mixture was
then warmed to room temperature and stirred overnight. After filtration, the solution
was concentrated, and the crude product was purified by flash chromatography to give
PA-B-PTX as a white solid (0.65 g, yield: 51.7%). Similarly, PA-H-PTX was obtained
as a white solid (yield: 69.4%).
2
.2.6 Synthesis of PEG-B-PTX
mPEG5k-OH (5.00 g, 1.00 mmol) was mixed with Et3N (2 mL) in 150 mL dry
THF. Methylsulfonyl chloride in 20 mL THF was then added dropwise under an argon
atmosphere with magnetic stirring in an ice bath. After stirring overnight at room
temperature, the mixture was filtered, and the solvent was evaporated and poured into
an excess of cold ether. The precipitate was isolated, washed with ether, and dried
1
under vacuum to obtain mPEG5k-OMs (4.50 g, yield: 90.0%) as a white solid. H
NMR (400 MHz, CDCl3) δ 4.43 – 4.31 (m, 2H), 3.78 – 3.49 (m, 458H), 3.36 (s, 3H),
3
.07 (s, 3H).

Journal Pre-proof
mPEG5k-OMs (4.50 g, 0.90 mmol) was dissolved in 30 mL DMF followed by the
addition of NaN3 (0.87 g, 13.4 mmol). The mixture was stirred at room temperature
for 3 days and then diluted with 300 mL CH2Cl2. The solution was washed with brine
three times and dried over anhydrous Na2SO4. After filtration, the solvent was
evaporated, and the resulting viscous oil was precipitated in excess of cold ether.
1
PEG5k-N3 (4.30 g. yield: 95.6%) was isolated and dried under vacuum. H NMR (400
MHz, CDCl3) δ 3.76 – 3.50 (m, 454H), 3.41 – 3.31 (m, 3H).
In a typical “click” reaction procedure, PEG5k-N3 (0.20 g, 0.04 mmol) and
PA-B-PTX (76 mg, 0.06 mmol) were added to a mixture of CuBr (5.76 mg, 0.04
mmol) and PMDETA (6.92 mg, 0.04 mmol) in 2 mL of DMF. The mixture was
degassed by three freezepumpthaw cycles, and then placed in a preheated oil bath
at 40 °C. After 72 h, the reaction was quenched upon exposure to air, diluted with
CH2Cl2, and passed through a neutral alumina column to remove the copper. The
eluent was concentrated and then precipitated into a mixture of cold ethanol and ether
(5:1) to remove the unreacted PA-B-PTX. The PEG-B-PTX was isolated by filtration
and dried under vacuum (0.16 g, 63.8%). PEG-H-PTX was obtained similarly as a
white solid (yield: 61.0%).
2
.3. Preparation of PEG-B-PTX and PEG-H-PTX micelles
Prodrug micelles were prepared using a nanoprecipitation method. PEG-B-PTX
(5 mg) or PEG-H-PTX (5 mg) were dissolved in 200 μL THF and added dropwise to
2
mL of deionized water with vigorous stirring. THF was removed under reduced
pressure after stirring for another 30 min. The volume-averaged diameters and zeta
potentials of the micelles were measured in triplicate using dynamic light scattering
(DLS), and the morphology of the micelles was observed by TEM.
2
.4. Critical micelle concentration (CMC) determination
The CMC of PEG-B-PTX or PEG-H-PTX was determined via the fluorescence
−6
method.[38] Briefly, Nile red (10 M, 50 μL) in CH2Cl2 was added into each vial.
The micelle solution (5 mL) free of THF was added at a series of predetermined
-
3
concentrations from 10 to 1 mg/mL. The vials were shaken overnight at 37°C in the
dark at a speed of 200 rpm. The fluorescence intensity at 620 nm (579 nm excitation)

Journal Pre-proof
of each solution was measured by a Molecular Device SpectraMax M2e reader. The
CMC was defined as the intersection of the two fitting lines of the plots of
fluorescence intensity versus the prodrug concentration.
2
.5. The micelles stability assessment
The PEG-B-PTX or PEG-H-PTX micelles (0.5 mM) were incubated in PBS (pH
.4, 10 mM), cell culture medium, and 10 % FBS at 37 °C with shaking (200 rpm).
7
The micelles’ stability in response to H2O2 was measured in PBS (pH 7.4, 10 mM) in
the presence of 200 μM H2O2. The micelles’ size change was tracked by DLS.
2
.6. In vitro H2O2 triggered PTX release
PEG-B-PTX or PEG-b-PTX micelles (1 mL, PTX equivalent concentration 1
mg/mL) were added to a dialysis tube (3500 MWCO) against 50 mL of PBS with or
without H2O2 (200 μM) and gently shaken at 37 ºC in a shaker at 150 rpm. At
predetermined time intervals, 100 μL of this solution was taken out for PTX
measurement by HPLC.
2
.7. Cell cytotoxicity assay
The cytotoxicity of PEG-B-PTX and PEG-H-PTX was determined by
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
cell
proliferation assay according to the manufacturer’s protocol with minor changes:[39]
U251 human glioma cancer cells, and MCF-7 breast cancer cells were briefly seeded
into 96-well plates at a density of 5000 cells per well and incubated for 18 h followed
by the addition of PMA (100 nM) and a further 6 h of incubation. After 48 h of
exposure to free PTX (dissolved in DMSO), PEG-B-PTX, or PEG-H-PTX at different
concentrations, 20 μL MTT (5 mg/mL) in PBS was added to each well and incubated
at 37 °C for 4 h until purple precipitates were visible. The solution in each well was
replaced with 100 μL of DMSO. The absorbance intensity of each well was then
determined at 562/620 nm using a Molecular Devices microplate reader. Each drug
concentration was tested in triplicate with three independent experiments.
2
.8. Cell apoptosis measurement
For apoptosis analysis, MCF-7 cells were seeded into 12-well plates at a density
4
of 10 cells per well and incubated for 18 h followed by the addition of PMA (100 nM)

Journal Pre-proof
with an additional 6 h of incubation. Free PTX, PEG-B-PTX, or PEG-H-PTX were
added at a dose of 5 μg PTX/mL and incubated for 48 h. The medium was removed,
and the cells were rinsed with PBS, detached by Trypsin, isolated, washed twice with
PBS, and resuspended in PBS. Cells were treated with Annexin V-FITC Apoptosis
TM
Detection Kit (Invitrogen) and analyzed using BD FACS Calibur flow cytometry
(Becton, Dickinson and Company, USA).
2
.9. In vivo pharmacokinetics analysis
Taxol (prepared according to the protocol), PEG-B-PTX, or PEG-H-PTX (10 mg
PTX-eq/kg) was injected intravenously into mice via the tail vein (n = 3). Blood
samples (50 μL) were collected via the orbital venous plexus of mice at predetermined
time intervals and then mixed with 0.1 M acetic acid (50 μL). The mixtures were
incubated at 37 °C overnight to facilitate the release of PTX. The next day, 1 mL of
methanol was added, thoroughly vortexed, ultrasonicated, and then centrifuged at
5
000 rpm for 5 min. The supernatants were taken and evaporated to dryness under
reduced pressure; 100 μLof acetonitrile was then added to re-dissolve the residue, and
the PTX content was measured by HPLC. At the experimental endpoint, animals were
sacrificed in accordance with the regulations of the Animal Care and Use Committee
of Zhejiang University.
2
.10. In vivo antitumor efficacy
Female BALB/c nude mice (68 weeks) were inoculated with MCF-7 cells (1 ×
7
1
0 cells per mouse) via subcutaneous injection into the right flank of mice. When the
3
inoculated tumors reached ~100 mm , the mice were randomized into four groups (n
=
6 per group) and treated with i.v. injection of 0.2 mL of PBS, Taxol, PEG-H-PTX,
or PEG-B-PTX at 10 mg/kg PXT-equivalent dose every two days for five times. The
length (L) and width (V) of the tumors, as well as the bodyweight of mice, were
monitored individually on alternate days. The tumor volume (V) was calculated using
3
2
the following formula V (mm ) = L(mm) × W (mm) × 0.5. The mice were sacrificed
on day 14 post-treatment, and the tumors were collected and weighed. The tumor
inhibition rate (TIR) was calculated by the equation: TIR = (mean tumor weight of the
control group − mean tumor weight of the treatment group)/mean tumor weight of

Journal Pre-proof
control group×100%.
.11. Histological assay
Tumor samples were washed in PBS, fixed with 4% neutral buffered
2
paraformaldehyde, embedded in paraffin, and cross-sectioned at a thickness of 10 μm.
The sections were stained with hematoxylin-eosin (H&E, Beyotime, China) and
observed under light microscopy.
2
.12. Statistical analysis
Statistical analysis used Excel and Origin. Comparisons were made using a
two-tailed, unpaired Student's t-test. P < 0.05 was considered statistically significant,
and all results are expressed as mean  standard deviation (s.d.).
3
3
. Results and discussion
.1. Synthesis and characterizations of PEG-B-PTX
The PEG-B-PTX was prepared as shown in Scheme 2. PEG-H-PTX with a
benzyl ester without the benzylboronic ester was synthesized as an unresponsive
counterpart for comparison. PEG-B-PTX was prepared from
-amino-4'-bromoacetophenone hydrochloride (compound 1) in six steps. Compound
was first protected with a t-butyloxycarbonyl group followed by reduction with
2
1
sodium borohydride and a Suzuki–Miyaura coupling reaction to obtain precursor 4.
After deprotection in DCM/TFA, compound 5 was reacted with PA-NHS to give
compound 6. Carboxylated PTX was obtained by esterification with succinic
anhydride. This was reacted with compound 6 to give PA-B-PTX. Finally,
PEG-B-PTX was obtained by PEG conjugation via a copper-catalyzed click reaction
(Scheme 2a). Similarly, the control prodrug, PEG-H-PTX, was synthesized using
2
-amino-1-phenylethanone hydrochloride as the starting material (Scheme 2b). The
1
H NMR spectra (Figs. 1a-1d) suggested complete conjugation of PEG-N3 and
PA-B-PTX or PA-H-PTX. Similar peaks were found for PEG-B-PTX and
PEG-H-PTX, except for the characteristic peak at 1.32 ppm belonging to the methyl
protons of benzylboronic ester. The structures of other intermediates were also
1
13
confirmed by H and C NMR spectra (Figs. S1S17) and MALDI-TOF mass spectra

Journal Pre-proof
(
Fig. S18). The molecular weights of PEG-B-PTX and PEG-H-PTX measured by
MALDI-TOF were 6403.5 and 6276.8 Da, respectively, which were consistent with
the theoretical values of 6404.0 and 6277.0 Da (Figs. 1e and 1f). The gel permeation
chromatography (GPC) results showed that PEG-B-PTX and PEG-H-PTX are both
single peaks with low polydispersity indices (PDI) of 1.06 and 1.05, respectively;
these data confirm the absence of free PEG and PA-B-PTX (Fig. 1g). The high purity
of PEG-B-PTX and PEG-H-PTX prodrugs was further confirmed by
high-performance liquid chromatography (HPLC) (Fig. S19).
Scheme 2. Synthesis of ROS-responsive PEG-B-PTX (a) and its unresponsive
control counterpart PEG-H-PTX (b). Reagents and conditions: i) (Boc)2O, Et3N,
THF, 0 ºC, 30 min, 40 ºC, 2 h; ii) NaBH4, MeOH, 0 ºC, 30 min; iii)
.
Bis(pinacolato)diboron, Pd(dppf)Cl2 CH2Cl2, KOAc, dioxane, 90 ºC, 12h; iv)
TFA/CH2Cl2 (1/2, v/v), RT, 6h; v) 4-pentynoic acid succinimide ester, Et3N, CH2Cl2,
RT, 1h; vi) PTX succinate, DCC, DMAP, CH2Cl2, 0 ºC, 30 min, RT, overnight; vii)

Journal Pre-proof
PEG-N3, CuBr, PMDETA, DMF, 40 ºC, 72 h.
Fig. 1. Structural characterization of the prodrugs PEG-B-PTX and
1
PEG-H-PTX. H NMR spectra of PA-B-PTX (a), PA-H-PTX (b), PEG-B-PTX (c),
and PEG-H-PTX (d) recorded in CDCl3. The MALDI-TOF MS spectra of
PEG-B-PTX (e) and PEG-H-PTX (f). GPC traces obtained for PEG-N3, PA-B-PTX,
PA-H-PTX, PEG-B-PTX, and PEG-H-PTX (DMF, 50°C, 0.8 mL/min) (g).
The PTX contents of PEG-B-PTX and PEG-H-PTX were 13.3% and 13.6% by
weight, respectively, as determined by HPLC. These amphiphilic prodrugs
self-assembled into micelles with similar sizes around 50 nm in diameter as
determined by DLS and confirmed by TEM (Fig. 2a). TEM images showed that the
nanoparticles were spherical and had a dark core indicating a micellar rather than
vesicular structure. The two prodrugs also had a similar critical micelle concentration
of around 30 μg/mL (Fig. S20). The low CMCs ensures the in vitro and in vivo
stability of their micelles. Notably, both PEG-B-PTX and PEG-H-PTX nanoparticle
solutions were stable for several months at 4 °C.

Journal Pre-proof
3
.2. ROS-responsive PTX release
Fig. 2. Micelle formation and drug release of PEG-B-PTX and PEG-H-PTX
prodrugs. (a) Size distribution patterns of PEG-B-PTX and PEG-H-PTX micelles
determined by DLS and their morphologies observed by TEM (inserted, scale bar =
2
00 nm); the prodrugs were dissolved in water at a concentration of 0.5 mM. (b) The
time-dependent size changes of PEG-B-PTX and PEG-H-PTX micelles (0.5 mM)
o
with or without 200 μM H2O2 at 37 C determined by DLS measurement. PTX release
profiles of PEG-B-PTX (c) and PEG-H-PTX (d) with or without 200 μM H2O2 at pH
7
.4 and 5.0.
The stability of micelles in 200 μM H2O2 in PBS (pH 7.4, 10 mM) was tracked
by DLS. The PEG-B-PTX micelles showed robust stability in the absence of H2O2,
but they gradually aggregated after treatment with H2O2; the sizes increased from 50
nm to 400 nm over 10 h and reached over 1 μm after 48 h (Fig. 2b). The formation of
aggregates was probably due to the oxidative cleavage of the intermediate
phenylboronic ester linkage resulting in shedding of the PEG corona and aggregation
of the released hydrophobic drugs (Scheme 1). In contrast, no size change of

Journal Pre-proof
PEG-H-PTX micelles was observed in either the presence or absence of H2O2.
The corresponding PTX release from PEG-B-PTX micelles was investigated
using dialysis bags in PBS under different pH and redox conditions to mimic different
physiological environments. PEG-B-PTX and PEG-H-PTX micelles were both very
stable and free of burst release with only 5% PTX release after 96 h in PBS at pH 7.4
without H2O2 (Fig. 2c and d). The acidic condition (pH 5.0) slightly promoted PTX
release from both micelles due to the acid-catalyzed hydrolysis of the ester bond [40].
As expected, H2O2 did not promote the PTX release from PEG-H-PTX micelles but
did dramatically accelerate the PEG-B-PTX release rate from 2% to 31% in 12 h and
6
5% in 96 h upon addition of 200 μM H2O2 at pH 7.4. Notably, the PTX release was
much faster if the micelles were incubated at pH 5.0 with H2O2 because of the
stronger oxidation potential of H2O2 in acidic solution [41]. Thus, the PEG-B-PTX
would remain stable in blood but display a fast PTX release in tumor
tissues—especially in the acidic tumor interstitium and lysosomes [42].
The ROS-trigged drug release of PEG-B-PTX may take place over two steps: (i)
oxidation of phenylboronic ester and subsequent 1,4-elimination [43] and (ii)
hydrolysis of succinate ester and release of PTX molecule. PEG-B-PTX was very
stable under normal physiological environments, and the esters could not be
hydrolyzed due to its steric hindrance and hydrophobicity. However, after the boronic
ester was oxidized, the resulting phenolate would undergo a spontaneous
1
,4-elimination to break the PEG-B-PTX conjugate with subsequent release of the
PTX succinate. After the removal of the hydrophobic phenylboronic acid pinacol ester,
the ester bond conjugated with PTX was further hydrolyzed due to its decreased steric
hindrance and the nearby hydrophilic environment. Thus, free PTX molecules were
successfully released.
3
.3. In vitro cytotoxicity
The cytotoxicity of these two prodrugs was compared on U251 human glioma
cancer cells and MCF-7 human breast cancer cells. Tumor cells are characterized by
high levels of ROS, which are responsive to various tumor microenvironments such
as proliferation-induced hypoxia and reoxygenation [44]. To mimic the high ROS

Journal Pre-proof
levels of tumor cells in vivo, an intracellular ROS stimulator
phorbol-12-myristate-13-acetate (PMA) [45] was preincubated with cells to elevate
the intracellular ROS level. The intracellular ROS was visualized by confocal laser
scanning microscopy (CLSM) using 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate
(H2DCFDA)—this probe is non-fluorescent but is oxidized to 2 ′ ,7 ′
-
dichlorodihydrofluorescein (DCF) upon oxidation by the intracellular ROS [46]. The
MCF-7 cells cultured with H2DCFDA (10 μM) showed low green fluorescence.
Strong green fluorescence was observed when cells were pretreated with PMA (100
nM) for 6 h, and the fluorescence declined to the control group level 12 h after the
treatment (Fig. 3a).
The PEG-B-PTX and PEG-H-PTX showed comparable cytotoxicity without
PMA stimulation with IC50 values of 3.87 μg/mL and 4.86 μg/mL on U251 cells, and
1
3.7 μg/mL and 9.47 μg/mL on MCF-7 cells, respectively (Figs. 3b and 3c). With
PMA stimulation, the cytotoxicity of PEG-B-PTX was greatly enhanced: The IC50
value was reduced by 11- and 14-folds on U251 and MCF-7 cells, respectively.
Notably, the cytotoxicity of PEG-B-PTX (IC50 = 0.35 μg/mL) was even higher than
PTX (IC50 = 1.08 μg/mL) upon PMA stimulation on U251 cell line. The PEGylated
prodrug reversed the drug resistance of U251 cells, efficiently entering the cells, and
PMA induced ROS elevation, triggering the fast release of PTX and enhancing the
cytotoxicity. PMA stimulation has no significant effect on the cytotoxicity of
PEG-H-PTX in either cell lines (Fig. 3b and 3c). We further studied the cell
apoptosis-inducing capability of both prodrugs by Annexin V-PI assay. The
percentage of cells in late apoptosis (both Annexin and PI-positive) was similar via
the treatment of PEG-B-PTX (14.38%) and PEG-H-PTX (13.17%) on cells without
PMA stimulation. However, once cells were pretreated with PMA, the percentage of
cells in late apoptosis increased to 30.75% after the treatment of PEG-B-PTX. This
was comparable to values in cells treated with free PTX (32.80%). In comparison, the
apoptosis-inducing ability of the unresponsive PEG-H-PTX was only slightly affected
by the elevated intracellular ROS level (18.68% vs. 13.17%). These findings

Journal Pre-proof
confirmed the crucial role of the ROS-responsive linker in the prodrug. The
ROS-triggered release of PTX-SA and its quick hydrolysis into free PTX in the cell
generated high cytotoxicity [47].
Fig. 3. ROS-dependent cell cytotoxicity. (a) Confocal microscopy images of
intracellular ROS generation in MCF-7 cells with or without 200 nM PMA for 6 or 12
h. All scale bars are 20 μm. Cell nuclei are stained by Hoechst 33342 and expressed as

Journal Pre-proof
blue. 2′,7′-Dichlorodihydrofluorescein (DCF)-induced by ROS is expressed as green.
(b, c) Effect of PMA-pretreatment on the cell cytotoxicity of PEG-B-PTX and
PEG-H-PTX to U251 cells (b) and MCF-7 cells (c). The cells were pretreated with
PMA (200 nM) for 6 h before the addition of the prodrug and free PTX. (d) The
apoptosis of MCF-7 cells was determined by Annexin V/propidium iodide double
staining. Cells were treated with PEG-B-PTX or PEG-H-PTX (PTX equivalent dose 5
μg/mL) for 48 h.
3
.4. Pharmacokinetics
The in vivo stealth property of PEG-B-PTX and PEG-H-PTX was subsequently
estimated by monitoring their blood clearance kinetics. Blood samples were collected
via the orbital venous plexus of mice at different intervals after a single intravenous
injection of PEG-B (H)-PTX or Taxol (PTX equivalent dose 10 mg/kg, n = 3). The
PTX content in plasma at different time intervals was determined by HPLC (Fig. 4).
Taxol was quickly cleared, and only 0.1% of the injected dose remained after 6 h. In
contrast, both PEG-B-PTX and PEG-H-PTX were slowly cleared; more than 10% of
the injected dose remained after 6 h. The blood clearance half-life (T1/2) and
area-under-curve (AUC) values of the formulations are listed in Table 1. The
PEG-B-PTX and PEG-H-PTX micelles exhibited superior pharmacokinetics to Taxol
with ~9.4 (10.4)-fold longer T1/2β and 4.8 (5.6)-fold larger AUC (P<0.05). Hence, the
PEG-B-PTX and PEG-H-PTX micelles had more favorable pharmacokinetics to
extravasate from the leaky tumor vasculature and can accumulate in tumor tissue [48].

Journal Pre-proof
Fig. 4. Blood clearance kinetics of PTX after a single injection of PEG-B-PTX,
PEG-H-PTX, and Taxol at a PTX equivalent dose of 10 mg/kg (n = 3).
Table 1. Blood half-life and area under the curve for different formulations of ICR
mice at a PTX-equivalent dose of 10 mg/kg.
Parameter
T1/2 alpha
T1/2 beta
AUC0~t
Unit
h
PEG-B-PTX
0.41 ± 0.03
PEG-H-PTX
0.41 ± 0.10
Taxol
0.10 ± 0.08
0.18 ± 0.05
29.63 ± 1.80
h
4.88 ± 1.61
5.44 ± 2.06
−1
−1
μg mL h
153.73 ± 29.20
140.11 ± 16.73
T1/2 alpha: the half-life of the distribution phase. T1/2 beta: the half-life of elimination
phases. Data are depicted as mean ± SD. Statistical significance: *P<0.05.
3
.5. In vivo antitumor efficacy
The in vivo antitumor activity of PEG-B-PTX micelles was compared with
PEG-H-PTX, Taxol, and PBS using an MCF-7 xenograft tumor model (Figs. 5a-5d).
The mice were randomly divided into four groups (n = 6) when the tumors reached
3
about 100 mm ; treatments were intravenously administered every two days for five
times. The mice were sacrificed one week after the last treatment, and the tumors
were extracted and weighted. Versus PBS, all treatments of PTX formulations led to
significant tumor growth inhibition. PEG-B-PTX exhibited the best antitumor activity
with a tumor inhibition rate of 89.4%, which is attributed to its favorable
pharmacokinetics and tumor-specific drug release. Unexpectedly, PEG-H-PTX

Journal Pre-proof
showed an even lower tumor inhibition rate (57.6%) than Taxol (68.5%, P < 0.05),
although PEG-H-PTX also has favorable pharmacokinetics. This result further
highlights the importance of the drug release profiles in affecting the antitumor
activity. The strong tumor inhibition capability of PEG-B-PTX was supported by the
histological analysis of tumor tissues—more apoptotic cells were found in tumors
treated with PEG-B-PTX than those treated with PEG-H-PTX or Taxol (Fig. 5e).
Moreover, no significant body weight loss was observed in the mice treated with
either PEG-B-PTX or PEG-H-PTX, but the mice treated with Taxol showed severe
body weight loss. The low toxicity of PEG-B-PTX and PEG-H-PTX prodrugs is
attributed to their improved pharmacokinetic profiles.
Fig. 5. In vivo antitumor activity. (a) The MCF-7 tumor volume changes in mice
treated with PBS, PEG-B-PTX, PEG-H-PTX, or Taxol (PTX-equivalent dose 10

Journal Pre-proof
mg/kg) with five replicate doses. (b) The average tumor weight of each group at the
end of the experiment. (c) The body weight variation of tumor-bearing mice during
the treatment (d) Images of the MCF-7 xenograft tumors of the mice at the end of the
experiment. (e) Representative histological features of the tumors 7 days after the last
treatment. Tissue paraffin sections were 10-μm thick. The tumor sections were stained
with hematoxylin–eosin and examined by light microscopy. Scale bars = 50 μm. Error
bars indicate s.d. (n = 6). *P < 0.05, **P < 0.01 (two-tailed Student’s t-test).
4
. Conclusion
We developed a novel ROS-responsive prodrug, PEG-B-PTX, by incorporating a
benzylboronic ester linker between PEG and PTX. The PEG-B-PTX prodrug has a
high drug loading content and can self-assemble into stable ~50 nm micelles. The
micelles are free of PTX release in the normal physiological environment but quickly
release PTX in tumor sites after ROS oxidation. As a result, the PEG-B-PTX prodrug
shows a more potent antitumor activity than an unresponsive counterpart PEG-H-PTX
prodrug and the clinical first-line drug Taxol. Moreover, the PEG-B-PTX prodrug
exhibits reduced systemic toxicity versus Taxol. Thus, this ROS-responsive prodrug
design is promising for cancer drug delivery. Importantly, this strategy may not be
limited to the delivery of PTX but can also be used to deliver other chemotherapeutic
drugs, bioactive peptides, and proteins.
Acknowledgements
We thank the National Natural Science Foundation of China (51833008, 21875211,
8
1672478, U1501243, and 51603181), the National Key Research and Development
Program of China (2016YFA0200301), and the Fundamental Research Funds for the
Central Universities of China (2019FZA4020).
Credit Author Statement
Chengyuan Dong, Quan Zhou, and Jiajia Xiang: Investigation, Methodology,
Formal analysis, Writing - Original Draft. Fusheng Liu: Supervision,
Visualization, Funding acquisition. Zhuxian Zhou and Youqing Shen: Writing -

Journal Pre-proof
Review & Editing, Supervision, Funding acquisition
Supplementary data
Supplementary material
References
[
1] J. Sandercock, M. Parmar, V. Torri, W. Qian, First-line treatment for advanced
ovarian cancer: paclitaxel, platinum and the evidence, Br. J. Cancer, 87 (2002)
15-824.
2] J. Crown, M. O'leary, The taxanes: an update, Lancet, 355 (2000) 1176-1178.
3] F.H. Wang, M. Porter, A. Konstantopoulos, P.C. Zhang, H.G. Cui, Preclinical
8
[
[
development of drug delivery systems for paclitaxel-based cancer chemotherapy, J.
Control. Release, 267 (2017) 100-118.
[4] Y.J. Zhang, Z.Y. Guo, Z.L. Cao, W.X. Zhou, Y. Zhang, Q.J. Chen, Y.F. Lu, X.L.
Chen, Q. Guo, C. Li, D.H. Liang, T. Sun, C. Jiang, Endogenous albumin-mediated
delivery of redox-responsive paclitaxel-loaded micelles for targeted cancer therapy,
Biomaterials, 183 (2018) 243-257.
[5] J. Xiang, B. Wu, Z. Zhou, S. Hu, Y. Piao, Q. Zhou, G. Wang, J. Tang, X. Liu, Y.
Shen, Synthesis and evaluation of a paclitaxel-binding polymeric micelle for efficient
breast cancer therapy, Sci. China Life Sci., 61 (2018) 436-447.
[6] J. Huang, B. Wu, Z. Zhou, S. Hu, H. Xu, Y. Piao, H. Zheng, J. Tang, X. Liu, Y.
Shen, Drug-binding albumins forming stabilized nanoparticles for efficient anticancer
therapy, Nanomed. Nanotechnol. Biol. Med., 21 (2019) 102058.
[7] R.W. Chakroun, F.H. Wang, R. Lin, Y. Wang, H. Su, D. Pompa, H.G. Cui,
Fine-Tuning the Linear Release Rate of Paclitaxel-Bearing Supramolecular Filament
Hydrogels through Molecular Engineering, ACS Nano, 13 (2019) 7780-7790.
[8] E. Miele, G.P. Spinelli, E. Miele, F. Tomao, S. Tomao, Albumin-bound
formulation of paclitaxel (Abraxane (R) ABI-007) in the treatment of breast cancer,
Int. J. Nanomed., 4 (2009) 99-105.
[9] I.H. Park, J.H. Sohn, S.B. Kim, K.S. Lee, J.S. Chung, S.H. Lee, T.Y. Kim, K.H.
Jung, E.K. Cho, Y.S. Kim, H.S. Song, J.H. Seo, H.M. Ryoo, S.A. Lee, S.Y. Yoon, C.S.
Kim, Y.T. Kim, S.Y. Kim, M.R. Jin, J. Ro, An open-label, randomized, parallel, phase
Ill trial evaluating the efficacy and safety of polymeric micelle-formulated paclitaxel
compared to conventional cremophor EL-based paclitaxel for recurrent or metastatic
HER2-negative breast cancer, Cancer Res. Treat., 49 (2017) 569-577.
[10] Z. Feng, G. Zhao, L. Yu, D. Gough, S.B. Howell, Preclinical efficacy studies of a
novel nanoparticle-based formulation of paclitaxel that out-performs Abraxane,
Cancer Chemother. Pharmacol., 65 (2010) 923-930.
[11] D.L. Stirland, J.W. Nichols, S. Miura, Y.H. Bae, Mind the gap: A survey of how
cancer drug carriers are susceptible to the gap between research and practice, J.
Control. Release, 172 (2013) 1045-1064.
[12] Q. Sun, Z. Zhou, N. Qiu, Y. Shen, Rational Design of Cancer Nanomedicine:

Journal Pre-proof
Nanoproperty Integration and Synchronization, Adv. Mater., 29 (2017) 1606628.
[
13] S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug
delivery, Nat. Mater., 12 (2013) 991-1003.
14] J. Wang, X. Sun, W. Mao, W. Sun, J. Tang, M. Sui, Y. Shen, Z. Gu, Tumor redox
heterogeneity-responsive prodrug nanocapsules for cancer chemotherapy, Adv. Mater.,
5 (2013) 3670-3676.
15] Z. Zhou, X. Ma, C.J. Murphy, E. Jin, Q. Sun, Y. Shen, E.A. Van Kirk, W.J.
[
2
[
Murdoch, Molecularly precise dendrimer-drug conjugates with tunable drug release
for cancer therapy, Angew. Chem. Int. Ed., 53 (2014) 10949-10955.
[16] J. Wang, S. Hu, W. Mao, J. Xiang, Z. Zhou, X. Liu, J. Tang, Y. Shen, Assemblies
of Peptide-Cytotoxin Conjugates for Tumor-Homing Chemotherapy, Adv. Funct.
Mater., 29 (2019) 1807446.
[17] Q. Zhou, S. Shao, J. Wang, C.H. Xu, J. Xiang, Y. Piao, Z. Zhou, Q.S. Yu, J. Tang,
X. Liu, Z. Gan, R. Mo, Z. Gu, Y. Shen, Enzyme-activatable polymer-drug conjugate
augments tumour penetration and treatment efficacy, Nat. Nanotechnol., 14 (2019)
7
[
99-811.
18] B.L. Sui, C. Cheng, M.M. Wang, E. Hopkins, P.S. Xu, Heterotargeted
Nanococktail with Traceless Linkers for Eradicating Cancer, Adv. Funct. Mater., 29
2019) 1906433.
19] J. Bhattacharyya, J.J. Bellucci, I. Weitzhandler, J.R. McDaniel, I. Spasojevic,
(
[
X.H. Li, C.C. Lin, J.T.A. Chi, A. Chilkoti, A paclitaxel-loaded recombinant
polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models,
Nat. Commun., 6 (2015) 7939.
[20] F.W. Zhang, S.Y. Zhang, S.F. Pollack, R.C. Li, A.M. Gonzalez, J.W. Fan, J. Zou,
S.E. Leininger, A. Pavia-Sanders, R. Johnson, L.D. Nelson, J.E. Raymond, M.
Elsabahy, D.M.P. Hughes, M.W. Lenox, T.P. Gustafson, K.L. Wooley, Improving
paclitaxel delivery: In vitro and in vivo characterization of PEGylated
polyphosphoester-based nanocarriers, J. Am. Chem. Soc., 137 (2015) 2056-2066.
[21] D.Q. Chen, G.Q. Zhang, R.M. Li, M.R. Guan, X.Y. Wang, T.J. Zou, Y. Zhang,
C.R. Wang, C.Y. Shu, H. Hong, L.J. Wan, Biodegradable, hydrogen peroxide, and
glutathione dual responsive nanoparticles for potential programmable paclitaxel
release, J. Am. Chem. Soc., 140 (2018) 7373-7376.
[22] X.D. Ma, E. Ozliseli, Y.Z. Zhang, G.Q. Pan, D.Q. Wang, H.B. Zhang, Fabrication
of redox-responsive doxorubicin and paclitaxel prodrug nanoparticles with
microfluidics for selective cancer therapy, Biomater. Sci., 7 (2019) 634-644.
[23] W. Zhang, X. Hu, Q. Shen, D. Xing, Mitochondria-specific drug release and
reactive oxygen species burst induced by polyprodrug nanoreactors can enhance
chemotherapy, Nat. Commun., 10 (2019) 1704.
[
24] R. Lin, A.G. Cheetham, P.C. Zhang, Y.A. Lin, H.G. Cui, Supramolecular
filaments containing a fixed 41% paclitaxel loading, Chem. Commun., 49 (2013)
968-4970.
25] X.L. Hu, G.H. Liu, Y. Li, X.R. Wang, S.Y. Liu, Cell-penetrating hyperbranched
4
[
polyprodrug amphiphiles for synergistic reductive milieu-triggered drug release and
enhanced magnetic resonance signals, J. Am. Chem. Soc., 137 (2015) 362-368.

Journal Pre-proof
[
26] C.E. Callmann, C.V. Barback, M.P. Thompson, D.J. Hall, R.F. Mattrey, N.C.
Gianneschi, Therapeutic enzyme-responsive nanoparticles for targeted delivery and
accumulation in tumors, Adv. Mater., 27 (2015) 4611-4615.
[27] Y. Shen, E. Jin, B. Zhang, C.J. Murphy, M. Sui, J. Zhao, J. Wang, J. Tang, M. Fan,
E. Van Kirk, W.J. Murdoch, Prodrugs forming high drug loading multifunctional
nanocapsules for intracellular cancer drug delivery, J. Am. Chem. Soc., 132 (2010)
4
259-4265.
[28] X. Hu, S. Zhai, G. Liu, D. Xing, H. Liang, S. Liu, Concurrent drug unplugging
and permeabilization of polyprodrug-gated crosslinked vesicles for cancer
combination chemotherapy, Adv. Mater., 30 (2018) 1706307.
[
29] L. Bildstein, C. Dubernet, P. Couvreur, Prodrug-based intracellular delivery of
anticancer agents, Adv. Drug Deliv. Rev., 63 (2011) 3-23.
30] J.J. Cheng, R. Tong, Paclitaxel-initiated, controlled polymerization of lactide for
[
the formulation of polymeric nanoparticulate delivery vehicles, Angew. Chem. Int.
Ed., 47 (2008) 4830-4834.
[
31] T.P. Szatrowski, C.F. Nathan, Production of large amounts of hydrogen peroxide
by human tumor cells, Cancer Res., 51 (1991) 794-798.
32] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by
[
ROS-mediated mechanisms: a radical therapeutic approach?, Nat. Rev. Drug
Discovery, 8 (2009) 579-591.
[33] J. Xiang, X. Liu, Z. Zhou, D. Zhu, Q. Zhou, Y. Piao, L. Jiang, J. Tang, X. Liu, Y.
Shen, Reactive Oxygen Species (ROS)-Responsive Charge-Switchable Nanocarriers
for Gene Therapy of Metastatic Cancer, ACS Appl. Mater. Inter., 10 (2018)
4
3352-43362.
[34] D. Zhu, H. Yan, X. Liu, J. Xiang, Z. Zhou, J. Tang, X. Liu, Y. Shen,
Intracellularly Disintegratable Polysulfoniums for Efficient Gene Delivery, Adv.
Funct. Mater., 27 (2017) 1606826.
[35] M. Ye, Y. Han, J. Tang, Y. Piao, X. Liu, Z. Zhou, J. Gao, J. Rao, Y. Shen, A
tumor-specific cascade amplification drug release nanoparticle for overcoming
multidrug resistance in cancers, Adv. Mater., (2017) 1702342.
[36] J. Noh, B. Kwon, E. Han, M. Park, W. Yang, W. Cho, W. Yoo, G. Khang, D. Lee,
Amplification of oxidative stress by a dual stimuli-responsive hybrid drug enhances
cancer cell death, Nat. Commun., 6 (2015) 6907.
[37] J. Li, A. Dirisala, Z. Ge, Y. Wang, W. Yin, W. Ke, K. Toh, J. Xie, Y. Matsumoto,
Y. Anraku, Therapeutic vesicular nanoreactors with tumor‐ specific activation and
self‐ destruction for synergistic tumor ablation, Angew. Chem. Int. Ed., 129 (2017)
1
4213-14218.
[38] Z. Zhou, X. Ma, E. Jin, J. Tang, M. Sui, Y. Shen, E.A. Van Kirk, W.J. Murdoch,
M. Radosz, Linear-dendritic drug conjugates forming long-circulating nanorods for
cancer-drug delivery, Biomaterials, 34 (2013) 5722-5735.
[39] Z. Zhou, Y. Piao, L. Hao, G. Wang, Z. Zhou, Y. Shen, Acidity-responsive
shell-sheddable camptothecin-based nanofibers for carrier-free cancer drug delivery,
Nanoscale, 11 (2019) 15907-15916.
[40] Z. Zhang, L. Mei, S.-S. Feng, Paclitaxel drug delivery systems, Expert Opin.

Journal Pre-proof
Drug Delivery, 10 (2013) 325-340.
41] X. Liu, J.J. Xiang, D.C. Zhu, L.M. Jiang, Z.X. Zhou, J.B. Tang, X.R. Liu, Y.Z.
[
Huang, Y.Q. Shen, Fusogenic reactive oxygen species triggered charge-reversal vector
for effective gene delivery, Adv. Mater., 28 (2016) 1743-1752.
[42] E.L. Jin, B. Zhang, X.R. Sun, Z.X. Zhou, X.P. Ma, Q.H. Sun, J.B. Tang, Y.Q.
Shen, E.V. Kirk, W.J. Murdoch, M. Radosz, Acid-active cell-penetrating peptides for
in vivo tumor-targeted drug delivery, J. Am. Chem. Soc., 135 (2012) 933-940.
[43] C. de Gracia Lux, S. Joshi-Barr, T. Nguyen, E. Mahmoud, E. Schopf, N. Fomina,
A. Almutairi, Biocompatible polymeric nanoparticles degrade and release cargo in
response to biologically relevant levels of hydrogen peroxide, J. Am. Chem. Soc., 134
(
[
2012) 15758-15764.
44] S. Prasad, S.C. Gupta, A.K. Tyagi, Reactive oxygen species (ROS) and cancer:
Role of antioxidative nutraceuticals, Cancer Lett., 387 (2017) 95-105.
45] Z. Deng, Y. Qian, Y. Yu, G. Liu, J. Hu, G. Zhang, S. Liu, Engineering
[
intracellular delivery nanocarriers and nanoreactors from oxidation-responsive
polymersomes via synchronized bilayer cross-linking and permeabilizing inside live
cells, J. Am. Chem. Soc., 138 (2016) 10452.
[46] Y. Yuan, C.J. Zhang, B. Liu, A photoactivatable AIE polymer for light‐
controlled gene delivery: concurrent endo/lysosomal escape and DNA unpacking,
Angew. Chem. Int. Ed., 54 (2015) 11419-11423.
[47] H.M. Deutsch, J.A. Glinski, M. Hernandez, R.D. Haugwitz, V.L. Narayanan, M.
Suffness, L.H. Zalkow, Synthesis of congeners and prodrugs. 3. Water-soluble
prodrugs of taxol with potent antitumor activity J. Med. Chem., 32 (1989) 788-792.
[48] Z. Zhou, X. Liu, D. Zhu, Y. Wang, Z. Zhang, X. Zhou, N. Qiu, X. Chen, Y. Shen,
Nonviral Cancer Gene Therapy: Delivery Cascade and Vector Nanoproperty
Integration, Adv. Drug Deliv. Rev., 115 (2017) 115-154.
Schemes, Figures andTable

Journal Pre-proof
Scheme 1. The chemical structure of the PTX prodrug, PEG-B-PTX, and it's
self-assembly into prodrug micelles for the tumor ROS-triggered drug release.

Journal Pre-proof
Scheme 2. Synthesis of ROS-responsive PEG-B-PTX (a) and its unresponsive
control counterpart PEG-H-PTX (b). Reagents and conditions: i) (Boc)2O, Et3N,
THF, 0 ºC, 30 min, 40 ºC, 2 h; ii) NaBH4, MeOH, 0 ºC, 30 min; iii)
.
Bis(pinacolato)diboron, Pd(dppf)Cl2 CH2Cl2, KOAc, dioxane, 90 ºC, 12h; iv)
TFA/CH2Cl2 (1/2, v/v), RT, 6h; v) 4-pentynoic acid succinimide ester, Et3N, CH2Cl2,
RT, 1h; vi) PTX succinate, DCC, DMAP, CH Cl , 0 ºC, 30 min, RT, overnight; vii)
2
2
PEG-N3, CuBr, PMDETA, DMF, 40 ºC, 72 h.

Journal Pre-proof
Fig. 1. Structural characterization of the prodrugs PEG-B-PTX and
1
PEG-H-PTX. H NMR spectra of PA-B-PTX (a), PA-H-PTX (b), PEG-B-PTX (c),
and PEG-H-PTX (d) recorded in CDCl3. The MALDI-TOF MS spectra of
PEG-B-PTX (e) and PEG-H-PTX (f). GPC traces obtained for PEG-N3, PA-B-PTX,
PA-H-PTX, PEG-B-PTX, and PEG-H-PTX (DMF, 50°C, 0.8 mL/min) (g).