An ROS-responsive and self-accelerating drug release nanoplatform for overcoming multidrug resistance

Article abstract of DOI:10.1039/c9cc00358d

An 'on-demand' drug release and ROS-responsive nanoparticle was prepared by chemically conjugating hydrophobic α-tocopheryl succinate to hydrophilic poly(ethylene glycol) via a thioketal linker. This nanoparticle encapsulated with doxorubicin and α-tocopheryl succinate exhibited remarkable efficiency in reversing multidrug resistance both in vitro and in vivo.

Full text of DOI:10.1039/c9cc00358d

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ROS-responsive and self-accelerating drug release nanoplatform
for overcoming multidrug resistance†
a
a
b
c
a
Xueming Lv, Yiyong Zhu, Hamidreza Ghandehari, Ao Yu* and Yongjian Wang*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
An ‘on-demand’ drug release and ROS-responsive nanoparticale only responsive to unique tumour microenvironment to realize
was prepared by chemically conjugating hydrophobic α-tocopheryl targeted drug delivery, but also have enhanced intracellular
succinate to hydrophilic poly(ethylene glycol) via thioketal linker. drug release profile.
This nanoparticale encapsulated with doxorubicin and α-
To achieve tumour targeted delivery and site-specific drug
tocopheryl succinate exhibited remarkable efficiency in reversing release, stimuli responsive nanosized DDSs have been
multidrug resistance both in vitro and in vivo.
developed in response to the stimuli signals in tumour
microenvironments, such as acidic pH, redox potential and
Cancer is still the leading cause of death all over the word.1
Although emerging anticancer strategies such as radiotherapy,
photothermal therapy and immunotherapy have been utilized
in clinic to treat cancer, conventional chemotherapy is still an
indispensable technique for cancer treatment. However, its
therapeutic efficiency is severely restricted by the development
of multidrug resistance (MDR) and the off-target toxicity of
chemotherapeutics. The mechanisms of MDR are rather
complicated and elusive, often include increased drug efflux,
decreased drug influx, enhanced DNA repair, altered cell cycle
6,7
reactive oxygen species (ROS). ROS including superoxide
-
anions (O
2
), hydroxyl radical (OH) and hydrogen peroxide
2 2
(H O
) are mostly generated in mitochondria due to the partial
8,9
reduction of oxygen. Excess production of ROS is related to a
series of pathological disorders, including inflammatory
10-12
diseases, cardiovascular diseases, diabetes and cancer.
Abnormal high intracellular ROS levels in cancer cells (up to 10
-5
×
1
0 M) are a distinguishable property from those in normal
-9
12
cells (around 20 10 M), which provide a unique target for
cancer therapy and a new direction to devise intelligent DDSs.
In contrast to the lysosomal acidic pH and intracellular high
concentration of glutathione which exist both in normal and
cancerous cells, the ROS level in cancer cells are higher than that
in normal tissues. This endows ROS-responsive DDSs possessing
better tumour selectivity. Although a variety of ROS-responsive
units, such as arylboronic ester, selenium, thioether, and
thioketal groups, have been integrated into DDSs for ROS-
triggered drug release, the current ROS-responsive DDSs are far
from optimal due to the fact that those developed ROS-
sensitive DDSs cannot release their payloads sufficiently under
×
2
,3
regulation and blocked cell apoptosis. Over the past decades,
numerous studies have been carried out to improve the
therapeutic efficacy of traditional anticancer drugs by
circumventing MDR. Nanotechnology is the most exploited
strategy to combat MDR due to its ability to bypass exposure of
4
,5
drugs to the membrane efflux transporters. Although the
nanosized drug delivery systems (DDSs) can specifically deliver
chemotherapeutics to tumour through the enhanced
permeability and retention (EPR) effect, premature drug release
during circulation and insufficient drug release inside the
tumour cells still hinders the application of DDSs in clinic.
Therefore, DDSs developed for overcoming MDR should be not
13,14
the inherent intracellular ROS level.
Several studies have been carried out to construct DDSs
capable of amplifying ROS signals to facilitate responsive
intracellular drug release. Incorporation of photo-sensitizers
a. Key Laboratory of Bioactive Materials, Ministry of Education, College of Life
15-
into DDS is one of the prevalent approaches to generate ROS.
Sciences, Nankai University, Tianjin 300071, China. E-mail:
yongjian_wang@nankai.edu.cn
Departments of Pharmaceutics and Pharmaceutical Chemistry, and of
1
7
However, external long period irradiation usually causes non-
b.
specific ROS elevation in both normal and cancer cells which
could give rise to lethal damage to healthy tissues. In contrast
to addition of external triggers, ROS-responsive and self-
accelerating DDSs by co-delivery ROS-inducing agents with
anticancer drugs have become a major focus and exhibit
Bioengineering, Utah Center for Nanomedicine, Nano Institute of Utah, University
of Utah, Salt Lake City, UT, USA
College of Chemistry, Nankai University, Tianjin 300071, China. E-mail:
c.
esr@nankai.edu.cn
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C9CC00358D
Scheme 1 Schematic representation of the ROS-responsive and
self-accelerating micelle.
remarkably improved therapeutic efficiency.18,19 α-Tocopheryl
succinate (α-TOS), a kind of vitamin E derivative, is a commonly
used ROS-inducing agent. α-TOS acts as a competitive inhibitor
of complex II in mitochondria respiratory chain, thus causes the
accumulation of high concentrations of ROS. Herein, we
hypothesize that encapsulating α-TOS into ROS-responsive
DDSs can self-induce the amplification of tumour intracellular
ROS signal and achieve efficient drug release.
Fig. 1 1H NMR spectra of TK linker, mPEG2k-TK, TPGS and
mPEG2k-TK-TOS (A-D). DLS analysis and TEM image of PTKTD
micelles (E, F). Scale bar: 200 nm. Colloidal stability of PTKTD
micelles in PBS and PBS containing 10% FBS (G). Cumulative
release profile of DOX in the presence of different
2
0
As a proof of concept, we developed a novel ROS-responsive
DDS encapsulated with both -TOS and DOX (defined as PTKTD)
which can selectively self-induce tumour cell ROS amplification
to facilitate drug release and overcome MDR. As shown in
Scheme 1, a small amount of α-TOS could be released from
PTKTD with the stimulation of tumour intrinsic ROS. The
released α-TOS would interact with mitochondrial respiratory
complex II to generate ROS, which in turn accelerates the
collapse of PTKTD micelles and release more α-TOS. This
positive feedback loop ensures an efficient and thorough
release of α-TOS and DOX to induce cell apoptosis. This novel
ROS-responsive and self-accelerating drug delivery system
demonstrated strong cell-killing ability against MCF-7/ADR cells
in vitro and conspicuous tumour growth inhibition capability in
vivo, which makes it a promising approach for cancer therapy.
In this study, a ROS-responsive and self-accelerating drug
release nanosystem (PTK) was synthesized by covalently
conjugating -TOS to mPEG2k through the ROS-cleavable
thioketal (TK) linker. The synthetic routes of PTK were shown in
scheme S1 (ESI†). The chemical structures of the intermediates
2 2
concentrations of H O (H).
represented the characteristic peaks of PEG and α-TOS,
respectively (Fig. 1C). The H NMR spectrum of Fig. 1D
confirmed the successful synthesis of PTK. The PTKTD micelles
were prepared by thin-film hydration method. The mean
diameter of the PTKTD micelles were 58.7 nm with a narrow
polydispersity (0.172) characterized by dynamic light scattering
1
(DLS) as shown in Fig. 1E. Meanwhile, the stability of PTKTD
micelle was also assessed by DLS. No obvious variation in the
particle size was observed for PTKTD micelles both in PBS and
PBS containing 10% FBS for 48 h, suggesting excellent stability
of PTKTD micelles (Fig 1G). Noncovalent intermolecular
2
1-23
interactions
between DOX and α-TOS, like hydrogen bond
and π-π stacking might contribute to the micellar stability (Fig
S2, ESI†). The morphology of PTKTD micelles were confirmed by
TEM (Fig. 1F) which showed a generally spherical morphology
with an average diameter of 20-25 nm. The average diameter
determined by TEM was smaller than that characterized by DLS
due to the shrinkage of TEM samples under dry condition. The
diameter and morphology of PTK and PTKT micelles were also
confirmed by DLS and TEM (Fig. S3 and S4, ESI†). DOX loading
content (DL%) of PTKTD micelles is 9.2% and the encapsulation
efficiency (EE%) is 87.6%, which were investigated with UV-vis.
1
and final product were characterized using H NMR (Fig. 1 and
1
S1, ESI†). The H NMR spectrums of Fig. 1A and 1B showed the
characteristic peak at 1.57 ppm which belongs to the -CH
protons of TK linker. The peaks at 3.64 ppm and 0.88 ppm
3
2
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Fig. 2 Cell viability of MCF-7/ADR (A) and MCF-7 (B) after being
treated with DOX, PTK, PTKD, PTKT and PTKTD for 48 h. The
experiment was repeated three times and data was expressed
as average ± SD, ****P < 0.0001.
To verify the ROS-responsive property of PTKTD micelles,
the in vitro DOX release profile of PTKTD micelle was studied. As
shown in Fig. 1H, less than 15% of DOX was released from the
PTKTD micelles without adding extra H O even after 84 h
2 2
incubation. In contrast, ~27% and ~38% of DOX were released
from the PTKTD micelles after incubation with 0.1 mM and 1
2 2
mM H O respectively, indicating the ROS concentration
dependent release profile of DOX and also suggesting the
rationale for the ‘self-accelerating release’ concept of the
PTKTD micelle. The ROS-responsiveness of PTK was further
1
verified by H NMR as shown in Fig. S5 (ESI†). Disappearance of
the characteristic peak at 1.57 ppm confirmed that TK was
efficiently cleaved by ROS.
The cytotoxicity of the free DOX and PTKTD micelles at
equivalent doses was evaluated using DOX-resistant human
breast adenocarcinoma cells (MCF-7/ADR) and human breast
adenocarcinoma cells (MCF-7) by MTT cell proliferation assay
(Fig. 2). PTK micelles or PTK micelles encapsulated with -TOS
(PTKT) or DOX (PTKD), respectively, were used as controls to
illustrate the efficiency of PTKTD in reversing MDR. PTK micelles
showed negligible growth inhibitory effect against both MCF-
Fig. 3 Tumour inhibition study. Relative tumour volume (A) and
body weight changes (B) of MCF-7/ADR tumour bearing mice
after treatment with saline, DOX, PTKT and PTKTD within 14
7
/ADR and MCF-7 cells at the experimental concentrations.
Nevertheless, PTKT micelles showed noticeable cytotoxicity
against MCF-7/ADR and low toxicity against MCF-7 cells at
days evaluation period, **P 0.01, ***P 0.001. Photograph
<
<
relatively high concentrations. It is reported that ROS of tumour tissues at the end of the treatment (C). H&E staining
concentration in MCF-7/ADR cells was approximately 4 times images of heart, liver, spleen, lung, kidney and tumour, and
that in MCF-7 cells.2 Therefore, much more α-TOS would be TUNEL staining images of tumour (200 magnification) (D).
4
×
released from PTKT micelles in MCF-7/ADR cells in contrast to
that in MCF-7 cells, which in turn accelerate the disintegration
cytotoxicity against MCF-7 cells than free DOX which may
benefit from the self-accelerating drug release profile, and the
synergistic effect of DOX and α-TOS.
of PTKT micelles to realize thorough release of α-TOS and
2
8
2
5
oxidative stress amplification. PTKD micelles did not show
noticeable cytotoxicity against MCF-7/ADR cells (Fig. 2A), which
may be caused by the insufficient drug release under the initial
relatively low ROS concentration in MCF-7/ADR cells. As
expected, the PTKTD micelles exhibited conspicuous
cytotoxicity against MCF-7/ADR cells due to the self-
accelerating drug release mode and the synergistic effect of
DOX and α-TOS, indicating the potent capability of the PTKTD
micelles to reverse MDR. As for MCF-7 cells (Fig. 2B), PTKTD
micelles showed remarkable cytotoxicity. Unlike most reported
work in literature that the cytotoxicity of drug loaded
nanomedicine against sensitive tumour cells is usually lower
than that of the free drug because of the delayed drug
To investigate ROS regenerating ability of different
formulations, DCFH-DA was used to monitor the intracellular
2
9
oxidative species. As shown in Fig. S6 (ESI†), the ROS level was
significantly elevated in cells treated with PTKT or PTKTD
micelles, while no obvious ROS variations were observed for
DOX, PTK and PTKD groups, suggesting excellent ROS inducing
ability of PTKT and PTKTD micelles.
The in vivo antitumor activities of free DOX and its different
formulations were evaluated in MCF-7/ADR tumour bearing
BALB/c nude mice. The mice were randomly divided into four
groups (n=6) and treated with saline, free DOX, PTKT and PTKTD
micelles, respectively, at an identical DOX dosage of 5mg/kg. All
formulations were intravenously injected into tail veins every
release,2
6,27
PTKTD micelles exhibited significant higher
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three days and tumour volume and body weight were measured There are no conflicts to declare.
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DOI: 10.1039/C9CC00358D
every two days during the 14 days treatment period. The
antitumor efficacy of different formulations was shown in Fig.
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Conflicts of interest
4
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A novel ROS-responsive and self-accelerating nanoplatform was fabricated for
circumventing multidrug resistance and enhancing tumour chemotherapy.