Self-Boosting Catalytic Nanoreactors Integrated with Triggerable Crosslinking Membrane Networks for Initiation of Immunogenic Cell Death by Pyroptosis

Article abstract of DOI:10.1002/anie.202004180

Synthetic polymer vesicles spur novel strategies for producing intelligent nanodevices with precise and specific functions. Engineering vesicular nanodevices with tunable permeability by a general platform without involving trade-offs between structural integrity, flexibility, and functionality remains challenging. Herein, we present a general strategy to construct responsive nanoreactors based on polyion complex vesicles by integrating stimuli-responsive linkers into a crosslinking membrane network. The formulated ROS-responsive nanoreactor with self-boosting catalytic glucose oxidation could protect glucose oxidase (GOD) to achieve cytocidal function by oxidative stress induction and glucose starvation, which is ascribed to stimuli-responsive vesicle expansion without fracture and size-selective cargo release behavior. The GOD-loaded therapeutic nanoreactor induced an immunostimulatory form of cell death by pyroptosis, which has the great potential to prime anti-tumor immune responses.

Full text of DOI:10.1002/anie.202004180

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Accepted Article
Title: Self-Boosting Catalytic Nanoreactor Integrated with Triggerable
Crosslinking Membrane Networks for Initiation of Immunogenic
Cell Death by Pyroptosis
Authors: Junjie Li, Yasutaka Anraku, and Kazunori Kataoka
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202004180
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Self-Boosting Catalytic Nanoreactor Integrated with Triggerable
Crosslinking Membrane Networks for Initiation of Immunogenic
Cell Death by Pyroptosis
Junjie Li,* Yasutaka Anraku, and Kazunori Kataoka*
Abstract: Synthetic polymer vesicles spur novel strategies for
producing biologically intelligent nanodevices with precise and
specific functions (e.g. nanoreactors). Engineering vesicular
nanodevices with tunable permeability by a general platform without
involving trade-offs between structural integrity, flexibility and
without involving trade-offs between structural integrity, flexibility
and functionality by a general platform remains challenging.
Polyion complex vesicles (PICsomes), based on electrostatic
self-assembly of charge-matched polypeptides, have been
developed as nanoreactor toward nanomedicine application.[2b, 3]
The covalent crosslinking of PICsomes membrane creates
hyperstable structure, while it simultaneously compromises
membrane permeability. Interestingly, the crosslinking density
would be a paramount parameter for membrane permeability.3d
However, permeability regulation through the density of
permanent crosslinking does not allow for achieving precise and
specific functions. A rationalized strategy for designing smart
nanoreactor is to integrate stimuli-responsive linkers into
crosslinking membrane network.[2e, 2g, 4]
functionality remains challenging. Here, we present
a general
strategy to construct responsive nanoreactors based on polyion
complex vesicles by integrating stimuli-responsive linkers into
crosslinking membrane network. The formulated ROS-responsive
nanoreactor with self-boosting catalytic glucose oxidation could
protect glucose oxidase (GOD) to maintain long-term activity to
achieve cytocidal function by oxidative stress induction and glucose
starvation, which is ascribed to stimuli-responsive vesicle expansion
without fracture and size-selective cargo release behavior. The
GOD-loaded therapeutic nanoreactor induced an immunostimulatory
form of cell death by pyroptosis, which has the great potential to
prime anti-tumor immune responses.
Synthetic polymer vesicles featured with hydrophilic cavity and
hydrophobic membrane enable innovative nanodevices design,
such as intelligent drug delivery systems, nanoswimmers,
nanoreactors, and artificial organelles and cells.[1] They adapt to
the surrounding environment with precise and specific functions.
A key prerequisite to function precisely is to endow the vesicular
membrane with tunable permeability, which should be without
accompanying impaired capability as a protective layer for the
encapsulated catalysts. Current strategies available for
enhancing membrane permeability include incorporating stimuli-
responsive polymers or channel proteins into membrane or
using intrinsically permeable polymers.[2] Despite permeability
enhancement in these designs, limitations remain, such as time-
consuming sample processing, organic solvents contaminations,
and vesicle structure damage. Furthermore, there is a barrier to
extend specific designs to other responsive systems. Thus,
engineering vesicular nanodevices with tunable permeability
Scheme 1. (a) Schematic illustration of the ROS-responsive GOD-loaded
therapeutic nanoreactor with self-boosting catalytic glucose oxidation due to
membrane permeability enhancement during ROS production for achieving
cytocidal function via pyroptosis-mediated immunogenic cell death followed by
glucose starvation (i) and oxidative stress induction (ii). (b) Schematic
illustration of vesicle swelling from membranal crosslinking density decreasing
and membranal hydrophobic-to-hydrophilic transition followed by H2O2-
triggered cleavage of hydrophobic thioketal linker into hydrophilic thiols.
[*]
Dr. J. Li, Prof. Dr. K. Kataoka
Innovation Center of NanoMedicine, Kawasaki Institute of Industrial
Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821,
Japan
Toward this concept, we arrived at glucose oxidase (GOD)-
loaded reactive oxygen species (ROS)-responsive PICsomes
Email: li-j@kawasaki-net.ne.jp, k-kataoka@kawasaki-net.ne.jp
Dr. Y. Anraku
Graduate School of Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Prof. Dr. K. Kataoka
Institute for Future Initiatives, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
E-mail: kataoka@ifi.u-tokyo.ac.jp
composed
of
poly([2-[[1-[(2-aminoethyl)thio]-1-
methylethyl]thio]ethyl]-α,β-aspartamide) (PATK) as polycation
segments and PEG-b-poly(α,β-aspartic acid) (PEG-b-PAsp) as
polyanion segments to validate the functionalities of the
nanoreactor (Scheme 1). Upon H2O2 exposure, gradual
cleavage of ROS-responsive linker occurred, and the vesicle
architecture evolved with stable swelling. Correspondingly,
membrane permeability increased remarkably due to membranal
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.202004180
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crosslinking density decreasing, membranal hydrophobic-to-
hydrophilic transition, and vesicle swelling. The efficiency of
catalytic glucose oxidation was anticipated to become high
followed by permeability enhancement. Importantly, vesicular
structure could maintain integrity without fracture during
transformation, which protected the catalyst GOD from harsh
environment for long-term activity. Meanwhile, GOD loaded
therapeutic nanoreactor could induce immunogenic cell death by
pyroptosis. To the best of our knowledge, this is the first
example of self-boosting catalytic nanoreactor with the capability
to initiate immunogenic cell death by pyroptosis.
Using electrostatic complexation as the driving force, self-
assembly of the charged polymers into PICsomes was facilely
performed via physically mixing of PATK and PEG-b-PAsp
solutions without using organic solvents, followed by vortex for 2
min.
After
covalent
cross-linking
by
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC), the stability could be
further improved. TEM characterization showed the spherical
vesicle structure and dynamic laser light scattering (DLS) results
revealed that narrow size distribution with a polydispersity index
(PDI) below 0.05 (Figure S7 and Figure 1a). To investigate the
architecture transformation, 5, 50, and 100 equivalents of H2O2
to the thioketal linker was added into PICsomes solutions. Time-
dependent DLS analysis revealed that before H2O2 addition, the
PICsomes exhibits an average diameter of about 95 nm. Upon
exposure to H2O2, the size increased steadily without loss of
narrow size distribution and culminated in a large plateau rather
than fast dissociation (Figure 1a). The intact vesicular structure
was verified by TEM observation (Figure S7). The long-term
capability to resist fragmentation should be ascribed to
incomplete breakdown of crosslinking membrane network,[6]
which is often utilized as a general strategy to construct stimuli-
responsive hydrogels for sustained drug release control.[7] It
should be noted that the size limitation of PICsomes swelling is
approximately 130 nm irrespective of H2O2 concentration (Figure
1a and Figure S8). Along with change in dimensions, PICsomes
can expand to 256 percent of original volume and 187 percent of
original surface area. The increased surface area would result in
loose arrangement of polymer chains, which should enhance
membrane permeability. Förster resonance energy transfer
(FRET) provided further insights into arrangement of polymer
chains during PICsomes transformation (Figure 1c and 1d). In
consistency with DLS results, FRET ratio declined significantly,
especially in the initial stage of Cy3- and Cy5-labeled PICsomes
transformation, indicating a loose membrane structure formation.
Then, PICsomes were explored to load hydrophilic cargoes,
which were simply added into solution in the process of mixing
of PATK and PEG-b-PAsp. Size-dependent release behavior
was observed (Figure 1b). For 150 kDa dextran, H2O2-triggered
vesicle expansion had no apparent effect on the release rate.
For 12 kDa PEG and free Cy3 dye, H2O2 accelerated the
release remarkably. Specifically, less than 10% of free Cy3 was
released within 6 hours, but more than 80% could be released
upon H2O2 stimulation for 6 hours. Besides the loose structure
from vesicle swelling, membranal crosslinking density
decreasing and membranal hydrophobic-to-hydrophilic transition
also account for accelerated release. The vesicle expansion
without fracture and size-selective cargo release behaviors
would lay a robust foundation for exploring and developing
PICsomes as therapeutic nanoreactor to encapsulate some
catalysts (e.g. enzyme) for running in the long-term.
Initially, ROS-responsive polycation, PATK, was designed
with
a ROS-cleavable linker (thioketal) in the side chain.
Quantitative aminolysis of poly(β-benzyl-L-aspartate) (PBLA) by
acetone-[bis-(2-amino-ethyl)-dithioacetal] to form PATK was
1
confirmed by H NMR (Figure S1-S3). On the other hand, PEG-
b-PBLA was transformed into polyanion, PEG-b-PAsp, by
hydrolysis of BLA units under alkaline conditions (Figure S2-S3).
The gel permeation chromatography (GPC) characterizations of
PBLA, PEG-b-PBLA, PATK and PEG-b-PAsp confirmed the
narrow MW distribution (Figure S4-S5). The ROS-cleavage of
hydrophobic thioketal linker into hydrophilic thiols was evaluated
for PATK incubated with H2O2 by 1H NMR (Figure S6).[5] The
degradation rate agreed well with H2O2 concentration as
evidenced by comparing the integrals of thioketal peak and the
signal of by-product acetone arose from cleavage reaction. In
this research, H2O2 was chosen as a kind of ROS to investigate
the related stimuli-responsive behavior.
Figure 1. Vesicle expansion and membrane permeability enhancement of
ROS-responsive PICsomes (5 mg/mL) incubated with 5 equivalents of H2O2 to
the thioketal linker. (a) Time-dependent DLS analysis of size and size
distribution (PDI) of PICsomes incubated with H2O2. After 300 hours, size
showed little change as indicated by black rectangle. (b) Quantification of 150
kDa dextran, 12 kDa PEG, and free sulfo-Cy3 release as a function of time.
Diagonal pattern indicates the addition of H2O2. (c) Time-dependent FRET
spectra of Cy3- and Cy5-labeled PICsomes incubated with H2O2 and (d)
quantification of corresponding FRET ratio.
Researches focus on GOD-loaded nanocarriers for cancer
treatment are quickly emerging.[8] Next, GOD-loaded PICsomes
(GOD@PICsomes) with 2.51% loading capacity were utilized to
validate the functionalities of the formulated therapeutic
nanoreactor. GOD encapsulation into PICsomes did not affect
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the vesicular structure and size (Figure S9). From fluorescence
correlation spectroscopy (FCS) analysis, the increased diffusion
time of Cy5-lebeled GOD (Cy5-GOD) loaded PICsomes
compared with free Cy5-GOD implied the successful
encapsulation into PICsomes (Figure S10). Glucose, a common
substrate of GOD, could be catalyzed to produce H2O2 in the
presence of oxygen. It should be noted that PICsomes already
amplify oxidative stress. Enzyme kinetic analysis with a
Michaelis–Menten model confirmed this (Figure 2c). The
catalytic efficiency of GOD decreased after encapsulating into
PICsomes based on a rising Michaelis constant (Km) from 23.54
mM to 61.32 mM, indicating that the affinity of the substrate for
GOD appears to decrease, possibly owing to the barrier effect of
membrane. But pretreating nanoreactor with H2O2 could recover
the affinity apparently (28.7 mM in Km).[2h, 2i]
had
a certain degree of membrane permeability to small
molecule demonstrated in our previous research,[3c] allowing
glucose to slowly cross membrane. To evaluate the
transformation behavior, the nanoreactor was incubated with
glucose followed by size and FRET measurement. At 24 hours
post incubation, Cryo-TEM characterization showed that H2O2
from catalytic glucose oxidation triggered the nanoreactor
expansion with diameter increasing from 78 ± 9 nm to 87 ± 10
nm, while had little effect on membranes thickness (17.5 ± 1.6
nm versus 18.6 ± 1.3 nm) (Figure 2a). Even after 1-week
incubation, the nanoreactor still maintained integrity with little
GOD release (< 5%) (Figure S11).
Furthermore, cytocidal function of nanoreactor was
investigated against 4T1 mouse breast cancer cells. A non-
responsive nanoreactor was constructed by encapsulating GOD
into previously synthesized PICsomes with similar loading
capacity (2.63%) (Figures S13), [3c] which was used as a control
group. IC50 value of the nanoreactor was 0.15 μg/mL compared
with 0.28 μg/mL for non-responsive nanoreactor, indicating
higher cytotoxicity (Figures 3a). A plausible reason is that
membrane permeability enhancement from ROS stimulation
during treatment further self-boosted the capability to produce
ROS for cytocidal activity (Figure 3b). Although the toxicity of
nanoreactor was lower than that of free GOD with IC50 value of
0.06 μg/mL, the nanoreactor could protect GOD to maintain
long-term cytocidal activity. We incubated 4T1 cells with free
GOD and nanoreactor at their IC50 concentrations, respectively.
After treatment, the culture medium was collected and
concentrated, followed by another treatment. The results
revealed that recycled nanoreactor maintained the cytocidal
activity efficiently even after 3 cycles while free GOD lost activity
quickly. A reasonable explanation is that GOD proteolysis from
proteinase released from dead cells would deactivate GOD.
Consistent with the cytotoxicity experiments, free GOD lost 60%
activity after incubation with proteinase K for 4 hours as detected
by glucose oxidase activity assay, while GOD loaded into
nanoreactor kept more than 95% activity.[9]
Figure 2. The formation and enzyme kinetic analysis of nanoreactor. (a) Cryo-
TEM images and (b) DLS analysis of vesicular nanoreactor before and after
treatment with 4.5 mg/mL glucose for 24 hours. (c) Kinetic parameters (Km
and Vmax) for GOD, GOD@PICsomes, and GOD@PICsomes pretreated with
5 equivalents of H2O2 to the thioketal linker for 24 hours (GOD@PICsomes w/
ROS).
It is worth mentioning that the thicker membrane could be
observed compared with that of previously synthesized 100-nm
PICsomes,[3b] which is beneficial to high-efficacy protection and
precise activation of catalysts. The nanoreactor expansion was
also evidenced by DLS and FRET measurements (Figure 2b
and Figure S12). The H2O2-triggered membrane permeability
enhancement would further increase the catalytic efficiency,
which endowed the nanoreactor with self-boosting capability to
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Figure 3. Cytocidal function of nanoreactor against 4T1 cells. (a) IC50 values
of GOD (1), GOD@PICsomes (2), and GOD-loaded nonresponsive
nanoreactor (3). (b) Cytotoxicity of recycled GOD and GOD@PICsomes from
old culture medium at their IC50 concentration. (c) DNA damage of 4T1 cell
treated with GOD@PICsomes at GOD concentration of 0.2 μg/mL.
than 85% LDH release using cell lysis as a positive control and
70% PI/Annexin V double positive cells verified the membrane
pore formation (Figure S17). Level of pro-inflammatory cytokine
IL-1β was increased in culture medium up to 20-fold (Figure
S18). We further analyzed the release and cell-surface
expression of immune stimulants, specifically HMGB1 and
calreticulin. Nanoreactor treatment increased calreticulin
expression more than 4-fold from flow cytometry analysis
(Figure 4b). HMGB1 concentration also increased to 4.3 ng/mL
compared with that of 0.1-0.2 ng/mL for PBS group based on
ELISA assay (Figure 4c). These pro-inflammatory cytokines and
immune stimulants from immunogenic cell death by pyroptosis
could potentiate antitumor immune responses, as just recently
highlighted in several studies.[11]
Taken together, this work provided a general platform to
engineer vesicular nanodevices with tunable permeability
without involving trade-offs between structural integrity, flexibility
and functionality by integrating stimuli-responsive linkers into
crosslinking membrane network. Given that varying polycations
have already been used to form PICsomes,[3] it is feasible to
extend application of this flexible strategy to other responsive
vesicles. We can tailor the responsive properties in on-demand
manner, for example, smart tumor microenvironment-responsive
nanoreactors design. Successful fabrication of reduction-
responsive PICsomes and its triggered expansion without
fracture affirmed this strategy (Figure S19). Based on high
oxidative stress environment and high glucose uptake in tumor
site, it is reasonable to design ROS-responsive GOD-loaded
nanoreactor for site-specific activation and amplification of
oxidative stress, avoiding the production of excessive ROS in
blood and normal tissue. However, mounting evidence indicates
that ‘‘one size fits all’’ microenvironment does not exist in tumor.
There is clearly an urgent need to construct a series of
responsive vesicles with similar physicochemical properties for
screening optimal nanoreactor. Furthermore, we found that
GOD-loaded PICsomes as therapeutic nanoreactor with self-
boosting catalytic ROS production capability and long-term
cytocidal function could induce an immunostimulatory form of
cell death termed as pyroptosis, which has the great potential for
priming anti-tumor immunotherapy and opens new avenues for
using GOD-loaded nanocarriers as a cancer treatment modality.
To understand the mechanism of cytocidal action, we first
evaluated the oxidative damage to cellular components (Figure
S14). 78% tail DNA appeared for nanoreactor-treated cells
compared with that of 2% for PBS as detected by Comet Assay,
indicating severe oxidative DNA damage (Figure 3c). Real-time
observation of cell morphology by confocal laser scanning
microscopy (CLSM) was also conducted. After treatment with
nanoreactor, the cells displayed distinctive morphology
characterized with large bubbles blowing from the plasma
membrane, which is a form of programmed cell death termed
pyroptosis distinguished from apoptosis (Figure 4a and Figure
S15).[10] The characteristic morphology could also be observed
in another two cell lines, MDA-MB-231 human breast cancer
cells and HT1080 human fibrosarcoma cells (Figure S16).
Acknowledgements
Figure 4. Nanoreactor-mediated immunostimulatory form of cell death by
pyroptosis at GOD concentration of 0.2 μg/mL. (a) Bright field images of 4T1
cells at 12 hours post-incubation with GOD@PICsomes. The white arrows
indicated the pyroptotic cell death characterized with large bubbles blowing
from the plasma membrane. (b, c) The level of immune simulants, calreticulin
(b) and HMGB1 (c), for 4T1 cells at 12 hours post incubation with
GOD@PICsomes.
The financial support based on Center of Innovation Program
(COI stream) (Project No. JPMJCE1305) from Japan Science
and Technology Agency (JST) is gratefully acknowledged. J.L.
thanks the financial support from JSPS International Research
Fellowship (ID: P17356; Prof. Kataoka as the host researcher)
and Ministry of Education, Culture, Sports, Science and
Technology, Japan (MEXT) (Project No. 20K20209).
Plasma membrane pore formation from pyroptosis causes
the release of intracellular contents, including lactate
dehydrogenase (LDH), pro-inflammatory cytokines (e.g. IL-1β
and IL-18), and some immune simulants (e.g. HMGB1). More
Keywords: Self-assembly • vesicles • nanoreactors • pyroptosis
• immunogenic cell death • immunotherapy
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Entry for the Table of Contents
A general platform to engineer vesicular nanodevices with tunable permeability based on responsive polyion complex vesicles was
provided. The formulated GOD-loaded therapeutic nanoreactor with self-boosting catalytic glucose oxidation due to membrane
permeability enhancement during ROS production could achieve cytocidal function via pyroptosis-mediated immunogenic cell death
followed by glucose starvation (i) and oxidative stress induction (ii).
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