ATP-Mediated Transient Behavior of Stomatocyte Nanosystems

Article abstract of DOI:10.1002/anie.201906331

In nature, dynamic processes are ubiquitous and often characterized by adaptive, transient behavior. Herein, we present the development of a transient bowl-shaped nanoreactor system, or stomatocyte, the properties of which are mediated by molecular intera

Full text of DOI:10.1002/anie.201906331

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Accepted Article
Title: ATP-Mediated Transient Behavior of Stomatocyte Nanosystems
Authors: Hailong Che, Jianzhi Zhu, Shidong Song, Alexander Mason,
Shoupeng Cao, Imke Pijpers, Loai Abdelmohsen, and Jan
van Hest
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201906331
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ATP-Mediated Transient Behavior of Stomatocyte Nanosystems
Hailong Che, Jianzhi Zhu, Shidong Song, Alexander F. Mason, Shoupeng Cao, Imke A. B. Pijpers, Loai K. E. A.
Abdelmohsen* and Jan C. M. van Hest*
Abstract: Dynamic processes are ubiquitous in nature, and often
characterized by adaptive, transient behavior, mediated by molecular
interactions. Here, we present the development of a transient bowl-
shaped nanoreactor system, also known as stomatocyte, of which the
properties are mediated by molecular interactions. In a stepwise
fashion, we couple motility to a dynamic process, which is maintained
by transient events; namely, binding and unbinding of the biomolecule
adenosine triphosphate (ATP). The surface of the stomatocyte
nanosystem is decorated with polylysine (PLL), and regulation is
achieved by addition of ATP. The dynamic interaction between PLL
and ATP leads to an increase in the hydrophobicity of the PLL-ATP
complex and subsequently to a collapse of the polymer; this causes a
narrowing of the opening of the stomatocytes. The presence of the
enzyme apyrase, which hydrolyzes ATP, leads to a decrease of the
ATP concentration, decomplexation of PLL and reopening of the
stomatocyte. The competition between ATP input and consumption
gives rise to a transient state that is controlled by the out-of-
equilibrium process.
such not in a transient fashion. Arguably, closest to transiently
regulated motile systems are the DNA-based molecular motors or
walkers.[12]
Here we describe such transient behavior in a stomatocyte
nanosystem which is mediated by the addition of the biomolecule
adenosine-5’-triphosphate (ATP). Our design relies on our previously
reported bowl-shaped polymer vesicle, or stomatocyte[13], which is
composed of poly(ethylene glycol)-block-polystyrene (PEG-b-PS)
block copolymers and loaded with catalytically active species. In order
to modulate the output of such stomatocytes by ATP, the stomatocyte
surface was decorated with polylysine (PLL). These PLL moieties
were introduced to participate in the dynamic non-covalent
complexation with ATP, to alter the size of the opening of the
stomatocytes. Considering the fact that ATP contains a hydrophobic
adenine group and four negative charges, we hypothesized that the
formation of PLL/ATP complexes would introduce hydrophobic
adenine groups along the PLL backbone. Thus, the opening of the
stomatocyte would then be blocked to prevent access of substrates
(for the nanoreactor) or fuel (for the nanomotor) in the stomatocyte,
leading to a decreased activity or speed, respectively. To introduce
transient behavior in the system binding of ATP to polylysine should
be counteracted. This can be achieved when potato apyrase is
present in the system, which hydrolyzes ATP into AMP, resulting in
disassembly and recovery of the original switched-on output.
Moreover, by refeeding ATP to the system, the transient behavior of
the stomatocytes could be reinstalled, endowing it with non-
equilibrium behavior (Scheme 1).
One of the most inherent and interesting features of living systems
is their adaptability to show complex behavior, in response to a variety
of signals. Among these behaviors, autonomous motion has been an
important source of inspiration for scientists who, over the years, have
created a variety of synthetic motor systems, imitating biological
motility.[1] For example, molecular motors[2], micro- and nanoscale
sized Janus motors[3], self-assembled polymeric motors[4], movable
tubules and rods[5] have been developed. Regardless of the excellent
performance of these motor systems, there is
a fundamental
difference in the way movement is regulated in synthetic and natural
systems. Cellular autonomous motion (e.g., vesicular transport and
motility)[6], displays adaptive features as a result of competing
transient activation and deactivation processes, which are governed
by enzyme-mediated energy input and consumption, and molecular
interactions. Such dynamic processes are also referred to as out-of-
equilibrium or dissipative; mimicking these behaviors in synthetic
systems has recently drawn much attention from the scientific
community.[7] Introducing transient behavior into synthetic molecular
or nanoscaled systems has been demonstrated for active materials
with unique properties such as dissipative fibres[8], transient peptide
hydrogels, vesicles or microcapsules[9], temporally programmed
“breathing” microgels and polymersomes[10], and non-equilibrium
molecular recognition and colloidal systems[11]. In synthetic motor
systems switchable behavior has been introduced, for example by
making nanomotors temperature responsive[3b]. Compared to the
above-mentioned transient systems, these nanomotors require a
second stimulus to switch back to their initial states and operate as
[a]
H. Che, J. Zhu, S. Song, Dr. A. F. Mason, S. Cao, I. A. B. Pijpers,
Dr. L. K. E. A. Abdelmohsen, Prof. J. C. M. van Hest,
Eindhoven University of Technology, Institute for Complex
MolecularSystems, P.O. Box 513 (STO 3.41), 5600MB Eindhoven,
The Netherlands
Scheme 1. (a) Schematic representation of the transient deactivation and
activation of a stomatocyte nanosystem mediated by ATP. (b) Chemical
structure of mPEG45-b-PS210, N3-mPEG45-b-PS215, PLL200, ATP, and AMP,
respectively.
E-mail: j.c.m.v.hest@tue.nl; l.k.e.a.abdelmohsen@tue.nl
Supporting information for this article is given via a link at the end of the
document.
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The morphology of the assemblies was characterized by
transmission electron microscopy (TEM). Polymer stomatocytes with
an almost closed opening (smaller than 5 nm) were observed (Figure
1a). Most importantly, grafting the PLL brush did not affect the size
and morphology of the stomatocytes, and the particles were stable for
at least six months at room temperature (Figure S4, Supporting
Information). These bowl-shaped structures with small nanopores
were further confirmed by scanning electron microscopy (SEM)
(Figure 1b) and cryo-TEM (Figure S5, Supporting Information).
Zeta potential (ζ) measurements were performed to indicate
whether PLL was successfully coupled to the surface of the
Figure 1. (a) TEM and (b) SEM images of PLL-modified stomatocytes. The red
arrows in the images indicate the opening of the stomatocytes. All scale bars
represent 200 nm. (c) Zeta potential changes of the ATP-complexed PLL-
stomatocytes as a function of time upon the addition of apyrase. The PLL-
stomatocytes were pretreated with ATP (62.5 μM, r=1). Experimental conditions:
0.5 mg mL-1 PLL-stomatocytes; MES buffer (5 mM, pH 6.5).
To demonstrate that PLL-ATP complexation indeed led to
hydrophobic collapse and therefore to the closure of the narrow
stomatocyte opening, we used this system to construct ATP-mediated
transient nanoreactors (Figure 2a). For this purpose, horseradish
peroxidase (HRP) as a model enzyme was encapsulated in the cavity
of the stomatocytes to generate nanoreactors. The hybrid
stomatocytes were purified by spin filtration, and size exclusion
chromatography (SEC) data confirmed the complete removal of
unencapsulated enzymes (Figure S8, Supporting Information). To
determine enzyme loading efficiency, after purification organic solvent
was added to the stomatocyte solution to reshape the stomatocytes
back into spherical polymersomes, thereby releasing encapsulated
enzymes (Figure S9, Supporting Information).[15] The released
enzymes were collected to analyze incorporation efficiency, which
was calculated to be 9.5% based on the bicinchoninic acid (BCA)
assay.
stomatocytes. After surface modification, the
ζ value of the
stomatocytes in a 2-(N-morpholino) ethanesulfonic acid (MES) buffer
solution (5 mM, pH 6.5) increased from -2.2 mV to 6.1 mV due to the
protonated primary amine groups at the stomatocyte surface (Figure
S7, Supporting Information). To confirm that ATP was able to complex
with PLL the change of the stomatocyte surface charge was studied
over a wide range of charge ratios (r), where r=(4ATP4-):(200PL200+).
With an increase in r, the ζ value of the samples witnessed a
continuous decrease from an original value of 6.1 mv (no ATP) to 1.2
mV (r=1). It is well known that the enzyme potato apyrase can
effectively hydrolyze ATP into AMP and two phosphate Pi species.[14]
Due to the decreased charge of AMP compared to ATP this will lead
to a much weaker interaction with PLL, and recovery of the initial
positive surface charge. As expected, the addition of apyrase resulted
in a gradual increase in the zeta potential of the ATP complexed
stomatocyte solution, which then remained constant over time (Figure
1c). Interestingly, we found that the zeta potential recovery rates of
the system are highly dependent on the concentration of apyrase.
Control experiments demonstrated that the addition of AMP only led
to a 0.4 mV surface charge change, which means that the interaction
between AMP and PLL is indeed very weak (Figure S7, Supporting
Information).
Figure 2. Adaptive stomatocyte nanoreactors. (a) Scheme of the ATP-
mediated responsive nanoreactors. HRP was encapsulated inside the cavity of
the stomatocyte, and DMB was utilized as a substrate to evaluate the enzymatic
reaction. (b) TEM image of HRP loaded PL-stomatocytes. Scale bar represents
200 nm. (c) Asymmetric flow field−flow fractionation (AF4) fractograms and
radius of gyration (Rg) of empty PLL-stomatocytes and HRP loaded PLL-
stomatocytes.
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Enzyme incorporation was further indicated by the black
aggregates at the bottom of the stomatocytes as observed by TEM,
and also by a combination of asymmetric flow field flow fractionation
(AF4) and static light scattering (LS) analysis, which showed a
significant decrease in radius of gyration (Rg) after enzyme
movement (Figure 4a). As with many dynamic processes in nature,
motility (at the nanoscale) is a critical mechanism for many biological
processes. Despite the great efforts to incorporate life-like behaviors
into artificial structures, integration of motion with dynamic processes
has so far remained elusive. Although previous work has shown that
temperature can be used to tune the velocity of the stomatocyte
nanomotors,[16] the way motion was regulated is perpetual.
Developing motile systems in which its function is coupled to a
dissipative process is an exciting way to create complex, biomimetic
systems that are inherently dynamic. Here, PLL-modified nanomotor
systems were constructed by encapsulation of platinum nanoparticles
(PtNPs) inside the stomatocyte; these particles are known to
decompose hydrogen peroxide (H2O2) into oxygen, driving the
nanomotor. TEM characterization confirmed the incorporation of
PtNPs in the cavity of the stomatocytes (Figure 4b, Figure S12 and
S13, Supporting Information). Static light scattering analysis
showed that after encapsulation of PtNPs, the Rg value of the
stomatocytes decreased from 151.5±9.8 nm to 136.8±13.4 nm, which
is another sign that PtNPs were successfully loaded in the cavity of
the stomatocytes (Figure 4c).
encapsulation
(Figure 2b and 2c, Table S2, Supporting
Information). HRP was used to catalyze the oxidation of 3,3’-
dimethoxybenzidine (DMB), and the product was monitored via the
gradual increase in absorbance at 492 nm. Different ATP levels gave
rise to different biocatalytic activity, and the more ATP was added, the
lower the enzymatic reaction, as shown in Figure 3a. In the absence
of ATP, the nanoreactors showed the highest activity. This
controllable catalytic behavior is attributed to the ATP-induced
collapse of the PLL chains on the surface of the stomatocytes, which
hinders diffusion of the substrate into the cavity. The degree of
collapse of the PLL chains is regulated by the concentration of ATP,
thereby leading to modulated, and even switched-off enzymatic
activity at the highest ATP concentration. Next, we sought to re-open
the closed “mouth” of the stomatocytes by apyrase. As shown in
Figure 3b, in the absence of apyrase, the ATP-gated stomatocyte
nanoreactors demonstrated hardly any activity and were in the “OFF”
state. However, in presence of apyrase, a noticeable increase in the
enzymatic reaction was observed, which is caused by the reopening
of the stomatocytes, allowing substrate diffusion into the cavity.
Moreover, higher concentrations of apyrase led to faster catalysis,
which means switching on the nanoreactors is dependent on the
amount of apyrase. Importantly, temporal control of nanoreactors with
out-of-equilibrium catalytic behavior was explored. When ATP was
repeatedly applied to the system in the presence of apyrase, the HRP
enzymatic reaction first stopped and subsequently recovered, which
was caused by the transient complexation between PLL and ATP,
highlighting the dissipative nature of our system (Figure S11,
Supporting Information).
Figure 4. (a) Schematic representation of ATP-regulated PtNP loaded
stomatocyte nanomotors. (b) TEM image of PtNP loaded PLL-stomatocytes.
Scale bar represents 100 nm. (c) Asymmetric flow field−flow fractionation (AF4)
fractograms and radius of gyration (Rg) of empty PLL-stomatocytes and PtNP
loaded PLL-stomatocytes.
Figure 3. (a) UV absorbance at 492 nm of the oxidation of DMB as a function
of time upon the addition of different concentrations of ATP. (b) Addition of
apyrase switches nanoreactors back to the ‘ON” state. Experimental conditions
in a and b: 0.5 mg mL-1 PLL-stomatocytes; MES buffer (5 mM, pH 6.5); [HRP]=5
U mL-1 and [DMB]=400 µM.
Next, we performed measurements of motion with nanoparticle-
tracking analysis (NTA) in the presence of ATP (motion analysis is
described in the Supporting Information). In the absence of H2O2,
the motors demonstrated Brownian motion (Video S1, Supporting
Information). As a control, upon applying H2O2, PLL-modified motors
in the absence of ATP demonstrated directional autonomous
After establishing the strong dependence of the activity of the
nanoreactors on ATP and the responsiveness of the system toward
apyrase, we then adapted this responsive behavior to autonomous
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movement (Video S2, Supporting Information), in line with previous
These results indicate that there is a reversible molecular complexing
process inside this system. ATP induced the formation of ATP/PLL
complexes to block the “mouth” of the stomatocytes, turning the
motors into an “OFF” state; subsequently, the system was taken over
by enzyme-mediated ATP depletion in turn, which turned the motors
into an “ON” state owing to the increase of the hydrophilicity of the
polymers, and the subsequent increased access of substrate to the
nanocavity of the stomatocytes.
15, 17]
reports on the motion of stomatocyte nanomotors.[4a,
Indeed,
increasing fuel concentration resulted in a transition of the motion
mechanism from diffusive to ballistic, as evident by the acquired
anomalous diffusion exponent α (alpha) values (Figure S17 and Table
S4, Supporting Information). Even though such PLL-modified motors
displayed slightly lower velocity compared to the unmodified ones,
which implies that the attachment of PLL on the surface of the
stomatocyte has only a limited effect on the movement of the motors
(Figure 5a). As shown in Figure 5b, the MSD of the stomatocyte
nanomotors experienced a significant decrease upon the addition of
ATP, and the more ATP was applied, the more the MSD decreased.
Upon increasing ATP levels, the velocity of the motors decreased
steadily from 35 μm/s (no ATP) to 7 μm/s (r=1), respectively, which
agrees remarkably well with what we observed for the ATP-modulated
nanoreactors. Furthermore, PLL-motors in absence of ATP showed
consistently a five times higher rate of motion than for the motors
exposed to the highest ATP concentration. When 62.5 μM ATP (r=1)
was added, the movement was reduced to levels close to Brownian
motion (Video S3, Supporting Information), indicating a near
complete closure of the opening. In addition, the measurement of the
x and y trajectories of the particles at different ATP concentrations
suggested that the motors could undergo a transition from directional
movement to Brownian motion, as evidenced by the observed linearity
of the MSD curves (Figure S19, Supporting Information ). The
formation of PLL/ATP complexes leads to collapsed hydrophobic
domains around the surface of the stomatocytes which thus hinders
the penetration of fuel into the nanocavity, reducing the speed of the
motors. Moreover, DLS results show that the increase in ATP
concentration causes the increase in apparent hydrodynamic size of
the motors (Figure S16, Supporting Information) –highlighting the
effective binding of stomatocytes to ATP and thus, reducing the speed
of these nanomotors. To demonstrate the importance of this
hydrophobic domain formation we employed a shorter polylysine
(PLL100). When this polymer was used to decorate the surface of the
stomatocytes, the velocity profiles did not noticeably change after
injection of ATP, even though the PL chains were completely
complexed with ATP (r=1) highlighting that the PLL length drastically
affects the modulation(Figure S20, Supporting Information ).
Finally, we investigated the ability of such nanomotors to transiently
respond, with their output being regulated by an external dynamic
process (Figure 6a). To fulfill this goal, potato apyrase was introduced
into the system to compete with PLL for the ATP that was added to
the nanomotors, by decomposing ATP to AMP, of which the latter is
unable to regulate the movement of the nanomotors (Figure S21,
Supporting Information). To trigger the adaptive movement, 62.5
μM ATP (r=1) was added to the nanomotor system in presence of
apyrase and the nanomotor movement was observed in real-time.
NTA showed that after the addition of ATP, two distinct regimes could
be observed. In the first regime, the motors spontaneously diminished
their motion from the original 35 μm s-1 to a minimum value of 7 μm s-
Figure 5. (a) MSD and velocity of Pt loaded stomatocytes (solid line) and PLL-
stomatocytes (dotted line) in the presence of H2O2 at different concentrations.
(b) MSD and velocity of PtNP loaded PLL-stomatocytes in the presence of ATP
at different concentrations. Please note: the extracted MSDs were used as a
qualitative indication on how active our motors are as a result of their transient
binding or unbinding to ATP.
To demonstrate control over this coupling of transient motor
function, we studied the dynamic regulation of the motors under
different apyrase levels (Figure 6c). Notably, in all situations, the
motors attained Brownian motion upon addition of ATP，followed by
a spontaneous increase in velocity until it leveled off to almost the
starting value. At the highest concentration of apyrase (0.15 U mL−1),
the velocity returned to the initial value after just 10 min. When a lower
enzyme concentration (0.1 U mL−1) was applied, the system reached
its approximately original state of motion after a longer time (around
15 min), which shows that increasing the enzyme concentration leads
to a faster recovery and a shorter lifetime of the temporal OFF state.
At the lowest apyrase level (0.05 U mL−1), the motors also rapidly
switched off movement, followed by a gradual speed increase but the
final velocity (22 μm s-1) of the motors was significantly lower than the
initial value, which was caused by the lower ATP hydrolytic rate.
1
(Figure 6b). The switched off state of the motors was however
transient and the motors slowly auto-accelerated back to their initial
state (second regime). The recovery process took much longer than
the deceleration process, which was completed within 30 seconds.
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These observations demonstrate that the biocatalytic pathway which
dissipates energy plays a crucial role in controlling the transient states
of the motors.
of the nanoreactors and speed of the nanomotors. Consumption of
ATP switches the system back to the initial condition. Moreover, the
temporal control over movement relies on the amount of ATP input
and the rate of ATP consumption, endowing the system with “life-like”
properties. This design is distinct from traditional responsive motors
that operate at a thermodynamic ground state. We expect that the
concept in this work could promote a novel class of artificial adaptive
nanosystems, guided by biological design principles.
Acknowledgements
The authors acknowledge the Dutch Science Foundation (VICI
grant), the ERC Advanced grant Artisym 694120, the Dutch Min-
istry of Education, Culture and Science (Gravitation program
024.001.035) and the European Union’s Horizon 2020 research
and innovation programme Marie Sklodowska Curie Innovative
Training Networks Nanomed, (No. 676137) for funding.
Conflicts of Interest
Figure 6. (a) Schematic illustration of the ATP-mediated adaptive stomatocyte
nanomotors. (b) Velocity of the nanomotors as a function of time on the addition
of ATP (62.5 μM) to a PtNP loaded PLL-stomatocyte solution in the presence of
different concentrations of apyrase. (c) Three cycles of an adaptive nanomotor
system upon the repeated addition of ATP (62.5 μM) showing the out-of-
equilibrium movement of the system. The arrows in b and c indicate the addition
of ATP.
The authors declare no conflict of interest.
Keywords: ATP-responsive • out-of-equilibrium • stomatocytes •
nanoreactors • nanomotors
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This article is protected by copyright. All rights reserved.

10.1002/anie.201906331
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
ATP was utilized to mediate the
transient behavior of the stomatocyte
nanosystems of which the “opening”
was regulated by competition between
ATP input and consumption. This
work presents a step toward the
design of man-made adaptive
nanosystems, to mimic and
H. Che, J. Zhu, S. Song, Dr. A. F.
Mason, S. Cao, I. A. B. Pijpers, Dr. L. K.
E. A. Abdelmohsen*, Prof. J. C. M. van
Hest*
Page No.– Page No.
Title: ATP-Mediated Transient
Behavior of Stomatocyte
Nanosystems
understand the dynamic behavior
found in nature.
This article is protected by copyright. All rights reserved.