Communication
Cite This: J. Am. Chem. Soc. 2018, 140, 7373−7376 pubs.acs.org/JACS
Biodegradable, Hydrogen Peroxide, and Glutathione Dual
Responsive Nanoparticles for Potential Programmable Paclitaxel
Release
Daiqin Chen,†,‡,§ Guoqiang Zhang,†,‡ Ruimin Li,†,‡ Mirong Guan,†,‡ Xueyun Wang,†,‡ Toujun Zou,†,‡
Ying Zhang,†,‡ Chunru Wang,† Chunying Shu,*,† Hao Hong,*,§ and Li-Jun Wan*,†,‡
†
Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, and Beijing
National Laboratory for Molecular Sciences, Beijing 100190, China
‡
University of Chinese Academy of Sciences, Beijing 100049, China
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§
Department of Radiology, Center for Molecular Imaging, University of Michigan, Ann Arbor, Michigan 48109-2200, United States
*
S Supporting Information
Downloaded via WEST TEXAS A & M UNIV on April 9, 2021 at 17:03:09 (UTC).
ABSTRACT: Reactive oxygen species (ROS) and gluta-
thione (GSH) dual responsive nanoparticulate drug
delivery systems (nano-DDSs) hold great promise to
improve the therapeutic efficacy and alleviate the side
effects of chemo drugs in cancer theranosis. Herein,
hydrogen peroxide (H2O2) and GSH dual responsive
thioketal nanoparticle (TKN) was rationally designed for
paclitaxel (PTX) delivery. Compared to other stimuli-
sensitive nano-DDSs, this dual responsive DDS is not only
sensitive to biologically relevant H2O2 and GSH for on-
demand drug release but also biodegradable into
biocompatible byproducts after fulfilling its delivering
task. Considering the heterogeneous redox potential
gradient, the PTX loaded TKNs (PTX-TKNs) might
first respond to the extracellular ROS and then to the
intracellular GSH, achieving a programmable release of
PTX at the tumor site. The selective toxicity of PTX-
TKNs to tumor cells with high levels of ROS and GSH
was verified both in vitro and in vivo.
T he past decades have witnessed the tremendous develop-
ment of nano-DDSs for cancer chemotherapy.1−3 A
concept has been widely accepted that ideal nano-DDSs should
be able to not only keep the payloads from any passive leakage
in the delivery process but also readily unload the cargoes in a
controlled release manner once arriving at the target sites.4−6 In
order to meet these criteria, researchers have proposed many
stimuli-responsive nano-DDSs for the targeted delivery and
site-specific release of various therapeutic cargos.7−9 Both
external (including light, magnetic field, ultrasound, etc.) and
internal stimuli (e.g., pH, temperature, enzyme, redox potential,
etc.) have been intensively utilized to trigger the smart nano-
DDSs.10−13 Among these reported triggers, redox potential has
attracted much attention in the development of nano-DDSs for
cancer theranosis due to the uniquely heterogeneous redox
potential gradient in the tumor site.14−16 The extracellular
matrix is oxidative as a result of overproduction of ROS, while
the intracellular cytoplasma is reductive because of a relative
high level of GSH in most tumor sites.17,18 There are many
ROS-sensitive moieties, including boronic esters/acids,19,20
© 2018 American Chemical Society
sulfides,21,22 thioethers,23,24 thioketals,25,26 selenium,27,28 tel-
lurium,29,30 proline oligomers,31,32 etc. Meanwhile, most GSH
responsive nano-DDSs contain disulfide groups.33,34 However,
only a few of these redox potential responsive nano-DDSs can
simultaneously respond to ROS and GSH, which compromise
the therapeutic efficacy. Consequently, researchers are enthu-
siastic in developing ROS and GSH dual responsive nano-
DDSs to improve their drug delivery performance.35,36
Although great progress has been made, there are still some
limitations that need to be overcome, such as the poor
sensitivity to ROS and GSH simultaneously at their biological
concentrations, the suboptimized biocompatibility and bio-
degradability of the nano-DDSs, the promiscuous fate of
byproducts after fulfilling their tasks, and so on.37−40
Herein, ROS and GSH dual responsive TKN was
synthesized, which could respond to H2O2 and GSH at the
biological level, producing the byproducts of biocompatible
acetone and lipoic acid (Figure S1). The drug release and cell
studies results validated that PTX-TKNs could specifically
release PTX in the presence of H2O2 and GSH, suggesting that
this nano-DDS might fulfill the programmable on-demand drug
release both extracellularly and intracellularly in cancer cells
(Scheme 1). The in vivo antitumor efficacy of PTX-TKNs on
PC-3 tumor bearing mice showed that PTX-TKNs could not
only significantly inhibit the growth of PC-3 tumors but also
greatly alleviate the side effects of PTX and improve the quality
of life thereof.
To guarantee the biocompatibility of the subsequently
biodegraded byproducts, we chose dihydrolipoic acid
(DHLA) as the monomer, which is the reductive product of
lipoic acid (LA), a FDA-approved antioxidant.41,42 The
presence of the two −SH proton peaks at 1.28 and 1.32 ppm
in the 1H NMR spectrum of DHLA verified the successful
reduction of lipoic acid (Figure S2). The ROS responsive
oligomer (o-DHLA) was synthesized through a condensation
reaction of DHLA and 2,2-dimethylpropane (DMP). The 1H
NMR spectrum clearly demonstrated the presence of a
thioketal group corresponding to the proton peak at 1.58
Received: November 13, 2017
Published: May 25, 2018
7373 DOI: 10.1021/jacs.7b12025
J. Am. Chem. Soc. 2018, 140, 7373−7376
Journal of the American Chemical Society
Scheme 1. Schematic Illustration of the ROS and GSH Dual
Responsive Nano-DDSa
a
The structure of the ROS and GSH dual responsive nano-DDSs,
containing both ROS responsive (purple) and GSH responsive
(green) motifs.
ppm (Figure 1a). Notably, there were still obvious proton peaks
at 1.28−1.32 ppm in o-DHLA (marked by red double stars in
Figure 1. (a) 1H NMR spectrum of o-DHLA (red star-marked peak
corresponds to the impurities). (b) MALDI-TOF spectrometry of o-
DHLA (red star-marked peak corresponds to the matrix rather than o-
DHLA). (c) 1H NMR spectra of o-DHLA, o-DHLA treated with 100
mM H2O2, lipoic acid, and acetone in CDCl3. (d) 1H NMR spectra of
o-DHLA before and after exposure to oxygen flow in CDCl3.
Figure 1a), indicating the existence of active sulfhydryl residues.
The matrix-assisted laser desorption/ionization-time-of-flight
mass (MALDI-TOF) spectrometry indicated that the oligomer
contains 15 units of DHLA (Figure 1b). As expected, the low
polymerization degree preserved active sulfhydryl residues and
facilitated the subsequent cross-linking to incorporate GSH
responsiveness in the final product. After treatment with H2O2,
an obvious signal was observed at 2.17 ppm corresponding to
acetone, a product of the oligomer cleavage (Figure 1c). The
other product, LA, was confirmed by the appearance of peaks
around 1.72 ppm. All the above results demonstrated that the
obtained oligomer was biodegradable, and most importantly, its
degradation products (lipoic acid and acetone) were quite
biocompatible and safe for clinical uses.43,44 The oligomer was
then cross-linked to form disulfide bonds and used for PTX
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Communication
delivery. The disappearance of the proton peaks of −SH in o-
DHLA suggested the formation of disulfide bonds after the
exposure to oxygen (Figure 1d). The cross-linked product was
characterized with gel permeation chromatography (GPC),
with MW of 41 290 (Figure S3). The drug loading content
(DLC) was determined by high performance liquid chromatog-
raphy (HPLC) to be 10.2% (Figures S4 and S5).
The transmission electron microscopy (TEM) images
demonstrated that the PTX-TKNs was ca. 250 nm (Figure
2a), which agreed well with the dynamic light scattering (DLS)
Figure 2. TEM images and size distribution of PTX-TKNs response to
H2O2 (a,b) and r-GSH (c,d), respectively. Scale bars in (a), inset in
(a), and (c) represent 500, 100, and 100 nm, respectively. NR release
(e) and dual-staged NR release (f) triggered with 100 mM H2O2 and
10 mM r-GSH.
results (Figure 2b). Both TEM images and size distribution
results showed that PTX-TKNs degraded into ca. 40 nm of
smaller nanoparticles after incubation with 100 mM H2O2
(Figure 2a,b). The ζ-potential changes also confirmed the
presence of degradation products after H2O2 treatment, as a
characteristic peak appeared in the negatively charged region
(Figures S6 and S7), and the population of PTX-TKNs
declined while the amount of their degradation byproducts (40
nm particles) increased with the elevation of H2O2 concen-
tration (Figures S7−S9). Notably, the PTX-TKNs were
sensitive to H2O2 as low as 100 μM (Figures S8 and S9), a
biologically relevant level of H2O2 (50−100 μM). GSH
treatment only led to slight swelling of PTX-TKNs, with the
diameter changing from 250 to 260 nm and the constant ζ-
potential of PTX-TKNs (Figures 2c,d and S10). The NR
release curve showed that only 21% drug release was observed
under the normal condition; while the drug release reached up
to 62% and 87% after treatment with GSH or H2O2 within 24
h, respectively (Figure 2e). The dual-stage responsive NR
release performance showed that the passive NR release almost
reached plateau within 4 h, then the NR release was greatly
accelerated when H2O2 was added, and a further expedition of
DOI: 10.1021/jacs.7b12025
J. Am. Chem. Soc. 2018, 140, 7373−7376
Journal of the American Chemical Society
NR release was observed once the addition of GSH at 3 h
postaddition (Figures 2f, S11, and S12). All these results
confirmed that this nano-DDS could respond to H2O2 and
GSH, triggering on-demand drug release correspondingly.
The ROS responsiveness of PTX-TKNs was evaluated on
PC-3 cells with an inherently higher level of ROS, and CHO
cells were selected as the control (Figure S13). After treatment
of 6.9 μM free PTX, the cell viabilities of PC-3 cells and CHO
cells fell to 63% and 4%, respectively, suggesting that free PTX
did more harm to CHO cells than to PC-3 cells (Figure 3a).
Figure 3. Cell viabilities of PC-3 cells and CHO cells incubated with
free PTX (a), TKNs (b), and PTX-TKNs (c) at a series of gradient
concentrations. (d) Cell viabilities of CHO cells and CHO cells
pretreated with GSH-OEt incubated with PTX-TKNs at a series of
gradient concentrations. n = 6; *p < 0.05, **p < 0.01. (e) Microscopic
images of CHO and PC-3 cells treated with TKNs (1 mg/mL) or
PTX-TKNs (13.8 μM, with respect to PTX). Scale bar = 20 μm.
While after incubating with PTX-TKNs (13.8 μM), the cell
viabilities of PC-3 cells and CHO cells were 30% and 90%,
respectively. No significant cell death was observed in these two
cells after treatment with TKNs (Figure 3b), as confirmed by
the microscopy images in Figure 3e. Then some CHO cells
were treated with glutathione reduced ethyl ester (GSH-OEt)
to elevate the intracellular GSH level, and GSH-OEt treatment
did negligible harm to CHO cells (Figure S14). After treatment
with 13.8 μM PTX-TKNs, the cell viabilities of the pretreated
and control CHO cells were 57% and 91%, respectively (Figure
3d). On the contrary, neither the non-ROS responsive nor the
non-GSH responsive nanoparticles posed selective cytotoxicity
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to PC-3 cells or GSH-elevated CHO cells (Figures S15 and
S16). All the observations indicated that PTX-TKNs posed
much higher selective cytotoxicity to PC-3 and GSH-elevated
CHO cells than that to normal CHO cells, most probably
resulting from the ROS and GSH triggered drug release from
PTX-TKNs.
Finally, we tested the anticancer efficacy of PTX-TKNs on
PC-3 tumor bearing mice. PTX-TKNs showed obvious
accumulation at the tumor site after intravenous injection at
24 h postinjection (Figure S17). The tumor size chart and
H&E slices clearly showed that both Taxol (free PTX) and
PTX-TKNs could effectively inhibit tumor growth, with the
inhibition rate of 77% and 65% on the 20th day, respectively
(Figure 4b,c). The body weight fluctuation (Figure 4a), the
Figure 4. Body weight fluctuation curves (a), tumor growth charts (b),
tumor H&E slices (c). Scale bar = 100 μm. Alanine aminotransferase
(ALT, d) and aspartate aminotransferase (AST, e) levels of mice
injected with saline, Taxol, and PTX-TKNs, respectively (n = 5); **p
< 0.01.
alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) levels of the mice treated with Taxol suggested obvious
side effects of free PTX to the mice; however, the mice treated
with PTX-TKNs did not show any abnormality. Considering
the selective cytotoxicity of PTX-TKNs to ROS-rich PC-3 cells
and GSH-elevated CHO cells rather than to normal cells, which
was verified previously in the in vitro assays, all these results
should probably be ascribed to the on-demand drug release
triggered from PTX-TKNs by high levels of ROS and GSH in
the heterogeneous redox potential tumor environment.
In conclusion, a H2O2 and GSH dual-responsive DDS was
designed for PTX effective delivery. This dual responsive
property could lead to on-demand cargo release in tumor
microenvironment due to the higher level of H2O2 and GSH,
which has been verified both in vitro and in vivo. This nano
DDS platform is biocompatible and biodegradable, with the
byproducts being lipoic acid and acetone. Moreover, other
chemotherapeutic agents or genes might also be selectively
delivered by this smart dual responsive carrier. Therefore, the
DOI: 10.1021/jacs.7b12025
J. Am. Chem. Soc. 2018, 140, 7373−7376
Journal of the American Chemical Society Communication

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■ AUTHOR INFORMATION
Corresponding Authors
Zhang, X.-Z. Biomaterials 2017, 128, 136−146.
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*hahong@med.umich.edu (28) Ma, N.; Li, Y.; Ren, H.; Xu, H.; Li, Z.; Zhang, X. Polym. Chem.
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Chunru Wang: 0000-0001-7984-6639 2015, 6, 2817−2821.
(30) Li, F.; Li, T.; Cao, W.; Wang, L.; Xu, H. Biomaterials 2017, 133,
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Li-Jun Wan: 0000-0002-0656-0936 (31) Lee, S. H.; Boire, T. C.; Lee, J. B.; Gupta, M. K.; Zachman, A. L.;
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ACKNOWLEDGMENTS
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7376 DOI: 10.1021/jacs.7b12025

J. Am. Chem. Soc. 2018, 140, 7373−7376