Therapeutic Vesicular Nanoreactors with Tumor-Specific Activation and Self-Destruction for Synergistic Tumor Ablation

Article abstract of DOI:10.1002/anie.201706964

Polymeric nanoreactors (NRs) have distinct advantages to improve chemical reaction efficiency, but the in vivo applications are limited by lack of tissue-specificity. Herein, novel glucose oxidase (GOD)-loaded therapeutic vesicular NRs (theraNR) are const

Full text of DOI:10.1002/anie.201706964

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Accepted Article
Title: Therapeutic Vesicular Nanoreactors with Tumor-Specific
Activation and Self-Destruction for Synergistic Tumor Ablation
Authors: Junjie Li, Anjaneyulu Dirisala, Zhishen Ge, Yuheng Wang,
Wei Yin, Wendong Ke, Kazuko Toh, Jinbing Xie, Yu
Matsumoto, Yasutaka Anraku, Kensuke Osada, and Kazunori
Kataoka
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201706964
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Therapeutic Vesicular Nanoreactors with Tumor-Specific
Activation and Self-Destruction for Synergistic Tumor Ablation
Junjie Li,† Anjaneyulu Dirisala,† Zhishen Ge,* Yuheng Wang, Wei Yin, Wendong Ke, Kazuko Toh,
Jinbing Xie, Yu Matsumoto, Yasutaka Anraku,* Kensuke Osada,* and Kazunori Kataoka*
Abstract: Polymeric nanoreactors (NRs) have distinct advantages to
improve chemical reaction efficiency, but the in vivo applications were
limited by lack of tissue-specificity. Herein, novel glucose oxidase (GOD)-
loaded therapeutic vesicular NRs (theraNR) are constructed based on the
diblock copolymer containing poly(ethylene glycol) (PEG) and
activity.[3] Enzyme-loaded cross-linked polymeric vesicular NRs have
been demonstrated to accumulate in tumor site and function as a
therapeutic NR for even four days post administration to convert a
model prodrug into a highly fluorescent product at the tumor site.[3b]
However, the reaction may unavoidably occur in normal tissues as long
as there exist NRs in main organs. Therefore, for cancer therapy, it is
highly desirable to engineer therapeutic NRs that can be specifically
activated at tumor site to maximize the therapeutic efficacy and
minimize the adverse side effects.
Polymeric vesicles obtained from the self-assembly of amphiphilic
block copolymers represent the most frequently used NRs, which
possess the properties simultaneously loading hydrophilic and
hydrophobic molecules within the aqueous inner cavity and
hydrophobic membranes, respectively.[4] Notably, as a nanocarrier,
drug release from the stimuli-responsive polymeric vesicles can be
triggered by the external or internal stimuli.[5] As NRs, the reaction
inside the vesicular space can also be adjusted by stimuli primarily
through controlling permeability of the vesicular membranes and
transportation of substrates and products.[6] However, rare stimuli-
responsive polymeric vesicles were reported as smart therapeutic NRs
particularly for in vivo applications.
copolymerized
phenylboronic ester or piperidine-functionalized
methacrylate (P(PBEM-co-PEM)). Upon systemic injection, theraNR keep
inactive in normal tissues. At tumor site, theraNR are specifically activated
by tumor acidity via improved permeability of the membranes. Hydrogen
peroxide (H2O2) production by the catalysis of GOD in theraNR increases
tumor oxidative stress significantly. Meanwhile, high level of H2O2 induces
self-destruction of theraNR releasing quinone methide (QM) to deplete
glutathione and suppress antioxidant ability of cancer cells. Finally,
theraNR efficiently kill cancer cells and ablate tumor via the synergistic
effect.
Nanoreactors (NRs) with a confined reaction space have attracted great
interests to improve chemical reaction efficiency because of their wide
applications in
a variety of fields such as enzyme catalysis,
polymerization, nanoparticle or organic synthesis, and artificial
organelles.[1] Among them, therapeutic NRs have been proposed in
recent years and explored to treat diseases through conversion of toxic
substances into nontoxic ones to eliminate or reduce the lesions to the
body, or in situ transformation of nontoxic prodrugs into therapeutic
compounds for cell or bacteria killing.[2] Notably, NRs frequently show
the distinct advantages to protect the encapsulated catalysts from the
environmental media, and thereby preserving and controlling the
Herein, as
a proof of concept to construct high-efficiency
therapeutic NRs for in vivo applications, we engineered multifunctional
vesicular NRs with tumor-specific activation and self-destruction for
synergistic cancer cell killing (Scheme 1). The diblock copolymers
[*]
J. Li, Y. Wang, W. Yin, W. Ke, Prof. Dr. Z. Ge
CAS Key Laboratory of Soft Matter Chemistry
Department of Polymer Science and Engineering
University of Science and Technology of China
Hefei, Anhui 230026, China
E-mail: gezs@ustc.edu.cn
Dr. A. Dirisala, Dr. K. Toh, Dr. J. Xie, Prof. Dr. K. Kataoka
Innovation Center of Nanomedicine, Kawasaki Institute of Industrial
Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821,
Japan
Dr. Y. Anraku, Prof. Dr. K. Osada
Graduate School of Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
E-mail: anraku@bmw.t.u-tokyo.ac.jp,osada@bmw.t.u-tokyo.ac.jp,
Dr. Y. Matsumoto
Department of Otorhinolaryngology and Head and Neck Surgery,
Graduate School of Medicine and Faculty of Medicine, The
University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan
Prof. Dr. K. Kataoka
Policy Alternatives Research Institute, The University of Tokyo 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-1709, Japan
E-mail: kataoka@pari.u-tokyo.ac.jp
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
Scheme 1. Schematic illustration of (A) self-assembly of PEG-b-P(PBEM-co-
PEM) to form GOD-loaded NRs (theraNR) and (B) the functioning mechanism
of theraNR at tumor site including tumor pH-activation, H2O2 production, and
QM release for synergistically killing cancer cells.
[†]
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10.1002/anie.201706964
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composed of poly(ethylene glycol) (PEG) and copolymerized
phenylboronic ester or piperidine-functionalized methacrylate
(P(PBEM-co-PEM)) were designed and optimized via changing the
molar ratios of PPBEM and PPEM, which could self-assemble into
vesicular structure in aqueous solution at pH 7.4. After encapsulating a
model enzyme, glucose oxidase (GOD), therapeutic NRs (designated
as theraNR) were constructed. In theraNR, PPEM and PPBEM
segments serve as tumor pH (pH 6.5-6.8) and hydrogen peroxide
(H2O2)-responsive hydrophobic segments, respectively. At normal
tissues of pH 7.4, theraNR maintain inactive state. After accumulation
in tumor tissues, PPEM segments turn out to be hydrophilic due to
protonation at tumor acidity, which endows the permeability of
theraNR membranes and allows transportation of the nutrient small-
molecule substances (glucose and oxygen). The accessibility of GOD
catalyzes the oxidation reaction to produce massive H2O2.
(FITC)-labeled GOD was used and the GOD loading content was
determined to be 4.7% via fluorescence intensity of FITC.
PEG-b-PPEM block copolymer has been demonstrated to exhibit
pH-responsive behavior with ultrasensitivity.[10] The pKa value of
PPEM segment in PEG113-b-P(PBEM67-co-PEM23) was determined to
be 6.95 via acid-base titration in aqueous solutions (Figure 1C). Thus,
when the pH values were decreased from pH 7.4 to pH 6.8, PPEM
segments turn out to be positively charged and hydrophilic. Next, pH-
responsive behaviors of the various vesicles were studied (Figure S2,
SI). The zeta-potentials of the vesicles increased with pH changing
from 7.4 to 6.8 due to the protonation of PPEM segments. With PPEM
contents increasing in the block copolymers, higher positive charges
were observed. PEG113-b-PPBEM80 and PEG113-b-P(PBEM67-co-
PEM23) vesicles showed high stability without morphological change
with pH decreasing to 6.8 most likely due to low PPEM content in the
Accompanied by the concentration reduction of the nutrient substances, block copolymers. Notably, PEG113-b-P(PBEM67-co-PEM23) vesicles
the oxidative stress in tumor tissue increases. The high concentration of
H2O2 conversely attacks PPBEM segments and induces self-destruction
of the vesicles to release quinone methide (QM) as by-products.[7] QM
possesses the capability to deplete intracellular glutathione (GSH), and
thus weakens the antioxidative capability of the cancer cells.[7b,8]
Increasing the oxidative stress and suppressing GSH can
synergistically kill cancer cells and inhibit tumor growth efficiently.
Initially, we designed the amphiphilic block copolymer, PEG-b-
P(PBEM-co-PEM), for self-assembly into vesicles with pH-
controllable permeability of the membranes while maintaining integrity
of the vesicles. PEG-based macroRAFT agent was used to
copolymerize PBEM and PEM monomers via reversible addition-
fragmentation chain transfer (RAFT) polymerization. A series of
PBEM/PEM ratios was designed and the final compositions were
determined by 1H NMR analysis (Figure S1, see Supporting
Information (SI)). The characterization of the final polymers were
summarized in Table S1 (SI). All the block copolymers were relatively
narrowly distributed with Mw/Mn in the range of 1.10-1.20. Next, the
block copolymers were explored to self-assemble in phosphate buffer
saline (PBS, pH 7.4) upon slow addition of PBS into tetrahydrofuran
(THF) solution of PEG-b-P(PBEM-co-PEM). All the block copolymers
can self-assemble into well-defined vesicle morphologies with the size
in the range of 100-200 nm as displayed by the transmission electron
microscope (TEM) and dynamic light scattering (DLS) analysis
(Figure S2, SI). TEM images show that spherical particles with high
contrast between the periphery and the center indicating typical
vesicular morphology. Typically, for PEG113-b-P(PBEM67-co-PEM23)
vesicles, the particles sizes were determined to be 119 ± 31 nm from
TEM images and the membrane thickness 21 ± 2.5 nm. DLS results
gave the particles size of 131 nm and low polydispersity index (PDI) of
0.116. Notably, although TEM images exhibited aggregated
morphology, DLS results showed well-dispersed nanoparticles in
aqueous solution indicating that the aggregation in TEM images likely
occurred during TEM sample preparation.[6c,6d,9]
displayed size of 122 ± 33 nm and membrane thickness of 22 ± 2.8 nm
as indicated from TEM images at pH 6.8, which were similar to those
at pH 7.4. In sharp contrast, PEG113-b-PPEM70 and PEG113-b-
Figure 1. (A) TEM images of theraNR at pH 7.4 and (B) theraNR at pH 6.8.
(C) Acid-base titration curve of PEG113-b-P(PBEM67-co-PEM23). (D)
Absorbance (abs, 370 nm) of the product (oxidative TMB) after cascade
reactions in theraNR or NC solution loading GOD (100 mU/mL) in the
presence of glucose (1 mg/mL), HRP (150 mU/mL), and TMB (100 µM)
outside the vesicles at pH 7.4 or pH 6.8. (E) H2O2 production of theraNR or
GOD (100 mU/mL GOD) in the presence of 1 mg/mL glucose at pH 7.4 or pH
6.8. (F) QM release from theraNR (100 mU/mL GOD) in the presence of 1
mg/mL glucose at pH 7.4 or pH 6.8. Mean ± s.d., n = 3.
Moreover, the interior cavity of the vesicles can be used to
encapsulate hydrophilic GOD. After encapsulating GOD, they were
washed with PBS to remove the unloaded free GOD. The size and size
distribution of the vesicles after loading GOD nearly maintained
constant (Figure 1A and Figure S3, SI). Fluorescein isothiocyanate
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P(PBEM65-co-PEM40) vesicles lost the integrity at pH 6.8 as observed
in the TEM images. According to our aim for tumor acidity activation
of the nanoreactors and protection of the encapsulated enzymes by the
vesicles for a period of time, GOD-loaded PEG113-b-P(PBEM67-co-
PEM23) vesicles were selected for further investigation due to the
suitable PPEM content, which were denoted as theraNR. Note that,
theraNR showed similar morphologies and particle sizes at pH 6.8 and
pH 7.4 (Figure 1A,B). The GOD-loaded PEG113-b-PPBEM80 vesicles
were used as a control of the nanocarrier without pH-responsiveness
(NC).
segments. However, theraNR did not produce any H2O2 at pH 7.4
(Figure 1E).
On the other hand, with H2O2 concentration increasing, high
concentration of H2O2 will conversely react with PPBEM segments of
theraNR and degrade the polymers, simultaneously releasing QM as
by-products. To evaluate the release profiles of QM, we incubated
theraNR in the presence of glucose and measured the amount of
produced 4-hydroxybenzyl alcohol (HA) as QM can be converted to
HA in aqueous solution without other nucleophiles (Figure 1F).[7] At
pH 6.8, approximately 60% of QM was released within 72 h, while
slight QM was released at pH 7.4. Further morphological observation
showed that theraNR lost the structural integrity and degraded
gradually to pieces of fragments at pH 6.8 after 72 h (Figure S5, SI)
whereas no morphological change was observed at pH 7.4 (Figure S6,
SI). NC control exhibited no H2O2 production and only little QM
release at both pH 7.4 and 6.8 (Figure S7, SI). Notably, as shown in
Figure 1E, theraNR did not show significant H2O2 concentration
decrease during QM release since only a small quantity of H2O2 was
consumed. Moreover, in the incubation medium of theraNR containing
glucose, we added GSH at the concentration of 1 mM. After incubation
Next, we further evaluated the pH-responsive permeability
variation of theraNR through the cascade reaction of glucose oxidation
by GOD to generate H2O2 and subsequent reaction with 3,3',5,5'-
tetramethylbenzidine (TMB) and horseradish peroxidase (HRP).[11]
Glucose, TMB, and HRP were localized in the exterior environment of
the vesicles, thus the permeability of the vesicles can be determined by
monitoring the characteristic absorbance of oxidized TMB.[12] TMB,
glucose, and HRP were added into theraNR solution at pH 7.4,
followed by adjusting pH to 6.8. Apparently, the color of theraNR
solution became blue and the absorbance at 370 nm increased rapidly
indicating high permeability of the vesicle membranes (Figure 1D).
However, the solution of NC at pH 7.4 did not show any color change
after pH was reduced to 6.8. Note that, as compared with the previous
pH-responsive NRs based on channel proteins,[12] this system showed
easy preparation and more sensitive response to slight pH change from
pH 7.4 to pH 6.8. Further activity measurements confirmed similar
maximal reaction velocities (Vmax) indicating that the encapsulated
GOD preserved comparable activity to that of free GOD at pH 6.8 and
the self-assembly process for encapsulation did not affect GOD activity
obviously (Figure S4). The quantification of released H2O2 from
theraNR demonstrated that H2O2 concentration increased to more than
0.3 mM within 1 h at GOD concentration of 100 mU/mL and glucose
of 1 mg/mL, which was slightly lower than that of free GOD at pH 6.8
likely due to slightly less efficacious H2O2 generation across theraNR
membranes and consumption by the decomposition of PPBEM
for 24
h at pH 6.8, the mixture was subjected to liquid
chromatography-mass spectrometric (LC-MS) analysis. The product
between QM and GSH (QM-GSH) can be detected indicating effective
reaction between QM and GSH (Figure S8, SI). Taken together,
theraNR shows ultra pH-sensitive activation of permeability with pH
values changing from 7.4 to 6.8. The nutrients of glucose and oxygen
can be converted into H2O2 efficiently and release QM from the
destruction of the vesicles for GSH depletion (Figure S9, SI).
Cancer cells usually possess deficient reactive oxygen species
(ROS)-eliminating systems, which are more sensitive to the elevated
oxidative stress than normal cells.[13] On the other hand, cancer cells
develop improved antioxidant systems in the tumoral oxidative
medium, for example, high concentration of GSH in cytoplasm,
thereby rendering them adaptive to the intrinsic oxidative stress in
tumor tissue. To evaluate the synergistic effect of theraNR on cell
viability against cancer cells through massive H2O2 production and QM
release, we first investigated the intracellular ROS and GSH
concentration at pH 6.8 in the medium containing 1 mg/mL glucose.
As compared with free GOD and NC, theraNR showed significant
cellular ROS elevation (Figure S10 SI), and intracellular GSH levels
were reduced to 50% and 20% after 24 h and 48 h incubation,
respectively (Figure S11, SI). TheraNR displayed potent cell killing
ability at pH 6.8 (Figure 2A and Figure S12, SI) and induced a high
cell death ratio with half maximal inhibitory concentration (IC50) of 87
mU/mL of GOD in theraNR at glucose concentration of 1 mg/mL
compared with 503 mU/mL of free GOD (Figure S13, SI). Reasonably,
compared with free GOD, theraNR showed similar H2O2 generation
ability at pH 6.8, but simultaneously released QM which suppresses the
antioxidant capability of cancer cells.[7b] Notably, PEG113-b-PPBEM80
and PEG113-b-P(PBEM67-co-PEM23) polymers without GOD showed
negligible cytotoxicity at the concentration of 1 mg/mL (Figure S14,
SI). Moreover, theraNR at pH 7.4 and NC at both pH 7.4 and 6.8 all
displayed low cytotoxicity due to no membrane permeability (Figure
S15, SI). Comet assay was further used to detect DNA damage at the
level of the individual cell via a pattern of DNA migration by the
Figure 2. (A) GOD concentration-dependent cytotoxicity of theraNRs and NCs
without (-) or with (+) glucose (1 mg/mL) at pH 6.8. Mean ± s.d., n = 4. (B)
Comet assay and (C) quantitation of tail DNA for control, GOD, NC, and
theraNR at GOD concentration of 100 mU/mL at pH 6.8. Mean ± s.d., n = 20.
***p < 0.005 (t-test).
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electrophoresis gels. Figure 2B,C revealed that theraNR severely
destroyed the cellular DNA as detected by high ratio of tail DNA ~
90% compared with ~ 40% of free GOD due to the synergistic effect of
theraNR on DNA damage of cancer cells.
after collecting the main organs of the mice at 48 h post injection of
NC and theraNR loading cypate-GOD displayed significantly stronger
fluorescence intensity at tumor sites than other organs indicating high
tumor-targeting efficiency of the nanoreactors (Figure S18, SI).
To evaluate the in vivo performance of theraNR, A549 tumor-
bearing mice were established. TheraNR could maintain high stability
in the serum-containing medium (Figure S16, SI). Blood circulation
test using cypate-labelled GOD (cypate-GOD) revealed long
circulation time of theraNR with half life of ~ 10 h upon intravenous
injection, which is similar to that of NC, whereas free GOD was
cleared from the body rapidly (Figure 3A). In vivo imaging system
(IVIS) was used to investigate the biodistribution of theraNR, which
showed that theraNR had significant tumor accumulation after 12 h
post-administration (Figure S17, SI). Ex vivo fluorescence images
In tumor tissues, the average glucose level is several µM/g tumor
tissue and displays heterogeneity,[14] which is the level to guarantee the
reaction under the catalysis of the nanoreactors. We first used an
intravital confocal microscope (IVRTCLSM) to investigate the
generation of H2O2 in tumor tissues (Figure S19A, SI). After 24 h post
intravenous injection of theraNR, 6'-o-pentafluorobenzene sulfonyl-
2',7'-difluorofluorescein (BES-H2O2) solution was injected into the tail
vein and the green fluorescence intensity in tumor was detected using
dorsal window chamber models due to the fluorescence turnon after
treatment by H2O2.[15] The fluorescence intensity increased a factor of
four quickly in tumor tissue compared with the PBS, GOD, and NC
groups indicating significantly improved H2O2 level in tumor after
treatment by theraNR (Figure 3B,C and Figure S19B,C, SI). Next, we
observed H2O2 and GSH levels directly in the histological cross
sections of the tumor tissue after 24 h and 48 h post intravenous
injection of theraNR. Upon intratumor injection of BES-H2O2 and
ThiolTracker™ Violet probe, the corresponding fluorescence
intensities revealed that tumor H2O2 level was improved to
approximately twenty times and GSH concentration was decreased to
about one sixth compared with the PBS control (Figure 3D and Figure
S20, SI). Notably, we could observe high concentration of H2O2 even
after 48 h in tumor tissue suggesting that theraNR still keep active.
Meanwhile, we also used IVRTCLSM to investigate H2O2 level in liver
and blood circulation upon treatment by theraNR. Moreover, after
intravenous injection of BES-H2O2, we did not observe any H2O2 level
increase in both liver and blood (Figure S21 and S22, SI). Therefore,
we can infer that theraNR are inactive in normal tissues at pH 7.4 and
work specifically in tumor tissue at pH 6.8.
The synergistic effect of theraNR for elevation of oxidative stress
and GSH depletion in tumor tissues was expected to finally suppress
the tumor growth efficiently. Next, the antitumor efficacy of theraNR
was investigated using NC, free GOD, and PBS as control groups. As
shown in the tumor growth profiles, theraNR efficiently inhibit the
growth of A549 tumors and even ablate the tumors completely after 27
days treatment (Figure 3E). In sharp contrast, in PBS group, the tumor
volume increased to over 12-fold than the original one. In free GOD
and NC groups, the tumor sizes were also increased to more than 10
times. The body weights of the mice maintain continual increase
during treatment indicated that low systemic toxicity of the therapeutic
systems (Figure 3F). Hematoxylin-eosin (H&E) and terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
staining showed large areas of cell apoptosis and tissue necrosis in the
tumor tissues treated by theraNR group (Figure S23, SI). These results
indicated potent antitumor capability of theraNR.
In summary, as a proof-of-concept, we constructed theraNR with
tumor-specific activation and self-destruction based on optimized
block copolymer, PEG-b-P(PBEM-co-PEM), for efficient in vivo
antitumor application. Proper composition of PPBEM and PPEM
endows the formed vesicles with ultra-sensitive pH-responsive
permeability while maintaining structural integrity, which makes the
vesicles specifically activated at tumor site. The reaction by the
Figure 3. (A) Plasma clearance profiles after i.v. injection of cypate-GOD,
cypate-GOD-loaded NC, and cypate-GOD-loaded theraNR. Mean ± s.d., n = 3.
(B) Observation of H2O2 distribution in A549 tumor tissues by IVRTCLSM. PBS
or theraNR was injected into the tail vein of A549 tumor-bearing mice. At 24 h
post injection, BES-H2O2 was injected into the tail vein of the tumor-bearing
mice. (C) Time-dependent relative fluorescence intensity (RFU) in the tumors
treated with PBS or theraNR. Mean ± s.d., n = 3. ***p < 0.005 (t-test). (D)
Quantitation of H2O2 and GSH levels in tumor tissue via analysis of integrated
optical density (IOD) after treatment by PBS or theraNR using BES-H2O2 and
ThiolTracker™ Violet probe, respectively. (E) A549 tumor growth profiles of
the mice treated with PBS, GOD, NC, and theraNR at GOD dose of 10 U per
mouse. Mean ± s.d., n = 5. ***p < 0.005 (t-test). (F) Body weight change of the
mice treated with PBS, GOD, NC, and theraNR.
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We gratefully acknowledge financial support from National Natural
Scientific Foundation of China (NNSFC) Project (21674104) and the
Fundamental Research Funds for the Central Universities
(WK3450000002).
Keywords: nanoreactors • polymersomes • cancer therapy • enzyme
delivery • membrane permeability
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10.1002/anie.201706964
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Layout 1:
COMMUNICATION
In vivo therapeutic nanoreactors
(theraNR): Glucose oxidase (GOD)-
Junjie Li, Anjaneyulu Dirisala, Zhishen
Ge,* Yuheng Wang, Wei Yin, Wendong
Ke, Kazuko Toh, Jinbing Xie, Yu
Matsumoto, Yasutaka Anraku,* Kensuke
Osada,* and Kazunori Kataoka*
loaded
vesicular
theraNR
are
engineered to be specifically activated
by tumor acidity and produce H2O2 for
improving the tumor oxidative stress.
High level of H2O2 induces vesicle
Page No. – Page No.
self-destruction
quinone methide
glutathione and suppress antioxidant
ability of cancer cells. The synergistic
effect of theraNR results in efficient
cancer cell killing and tumor ablation.
and
release
to deplete
of
Therapeutic Vesicular Nanoreactors
with Tumor-Specific Activation and
Self-Destruction
Tumor Ablation
for
Synergistic
This article is protected by copyright. All rights reserved.