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Transient control of lytic activity via a
non-equilibrium chemical reaction system†
Cite this: Nanoscale, 2024, 16, 6442
Published on 02 March 2024. Downloaded on 10/19/2024 10:17:29 PM.

Received 28th December 2023,
Accepted 29th February 2024

Kohei Sato, *‡§a Yume Nakagawa,‡a Miki Mori,a Masahiro Takinoue
Kazushi Kinbara *a,c

a,b,c

and

DOI: 10.1039/d3nr06626f
rsc.li/nanoscale

The development of artiﬁcial non-equilibrium chemical reaction

phospholipid vesicles encapsulating ﬂuorescent dyes and redsystems has recently attracted considerable attention as a new

blood cells as cell models, we demonstrate that the developed

type of biomimetic. However, due to the lack of bioorthogonality,
such reaction systems could not be linked to the regulation of any

chemical reaction system is applicable for transient control of theresulting lytic activity.

biological phenomena. Here, we have newly designed a non-equilibrium reaction system based on oleﬁn metathesis to produce the
Triton X-mimetic non-ionic amphiphile as a kinetic product. Usinga

School of Life Science and Technology, International Research Frontiers Initiative,
Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501,
Japan. E-mail: ksato@kwansei.ac.jp, kkinbara@bio.titech.ac.jpbDepartment of Computer Science, International Research Frontiers Initiative, Tokyo
Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japanc
Living Systems Materialogy Research Group, International Research Frontiers
Initiative, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama
226-8501, Japan† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d3nr06626f‡ These authors contributed equally to this work.
§ Present address: Department of Chemistry, School of Science, Kwansei Gakuin
University, 1 Gakuen Uegahara, Sanda-shi, Hyogo 669–1330, Japan.

Kohei Sato received his PhD fromthe University of Tokyo in 2014
under
the
supervisionofProfessor Takuzo Aida. He thenconducted postdoctoral researchin the group of Professor SamuelI. Stupp at Northwestern
University. In 2018, he joined
the group of Professor Kazushi
Kinbara as an assistant professor. In 2023, he started his
independent career as an associate professor in the Department
Kohei Satoof Chemistry at Kwansei Gakuin
University. His current research focuses on the development of bioactive supramolecular materials.

6442 | Nanoscale, 2024, 16, 6442–6448

Introduction
The development of life-like architectures has long been theultimate goal in organic chemistry.1–3 To date, remarkableadvances in synthetic and supramolecular chemistry haveenabled the construction of complex molecular assemblies
reminiscent of natural biomacromolecules such as proteins4–15
and nucleic acids.16–22 More recently, the development of artificial systems that mimic the non-equilibrium chemical reactions in living organisms, known as systems chemistry, has
attracted considerable attention as a new type of
biomimetic.23–32 Furthermore, such reactions have been
associated with functional outcomes, for example, drug
release,33,34 modulation of chemical reactions,35,36 and control

Yume Nakagawa was born inMie, Japan in 1999. She completed her undergraduate course
from the Tokyo Institute ofTechnology and is currently continuing her research as a postgraduate student in the Kinbara
group. Her research interests are
non-equilibrium chemical reactions, particularly in Systems
Chemistry.
Yume Nakagawa

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Nanoscaleof the microscopic movement of colloids.37 However, to thebest of our knowledge, such reaction systems have never been
involved in interacting directly with biomolecules to regulate
any biological phenomena. Undoubtedly, non-equilibrium
chemical reaction systems are highly advantageous for theirpromising potential to precisely and temporally control biological activities. However, conventional systems chemistry was
mostly based on the reactions of polar functional groups,38
includingcarboxylic
acids
for
carbodiimide-fueledreactions,39,40 amines for imine formation,41,42 and thiols forthiol–ester and thiol–disulfide exchange reactions.43–45
Because such polar functional groups are abundant in biomolecules, these reactions would most likely show interference
under physiological conditions.
Recently, Fletcher and coworkers have developed non-equilibrium chemical reaction systems based on the ruthenium-catalyzed cross-metathesis of hydrophilic and hydrophobic
alkenes.46 Here, we took advantage of the bioorthogonality ofthe metathesis reaction47–50 and sought to introduce a newmolecular design strategy into Fletcher’s system to promote
direct interaction between the resulting product and biomolecules. The chemical structure of a well-known commercial
synthetic surfactant, Triton X (Fig. 1), provided us with a clue.
Triton X is a non-ionic amphiphile consisting of a hydrophobic
aliphatic tail, an aromatic ring, and a hydrophilic poly(ethylene
glycol) (PEG) chain, known for its high propensity to interact
with lipid molecules to disrupt biomembranes while preventing
protein denaturation.51,52 Therefore, we designed a non-equilibrium chemical reaction system based on olefin metathesis to
produce a Triton-mimetic amphiphile as a kinetic product.
Through this study, we expected to establish a proof-of-concept
system to realize the temporal control of biological activity synchronous to an artificial non-equilibrium chemical reaction. In
particular, we focused on controlling the interaction with cell
membranes (Fig. 1), the vital components of life.3,53,54

Masahiro Takinoue is a biophysicist studying artificial cellengineering and molecular computing based on DNA nanotechnology. He received a B.Sc.
in Physics in 2002, a M.Sc. in
Physics in 2004, and a Ph.D. in
Physics in 2007 from The
University of Tokyo (UT), Japan.
After serving as a postdoctoral
fellow at Kyoto University and a
project assistant professor at UT,
he served as an associate proMasahiro Takinoue
fessor at Tokyo Institute ofTechnology (Tokyo Tech), Japan (2011-2022). He has been a full
professor at the Department of Computer Science and Living
Systems Materialogy (LiSM) Research Group at Tokyo Tech
(2022-).

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Communication

Fig. 1 Schematic illustrations of the chemical structure of Triton X-114
and a non-equilibrium chemical reaction system that produces the
Triton-mimetic amphiphile as a kinetic product through oleﬁn metathesis. The amphiphile was designed to interact with cell membranes to
induce their disruption in a transient manner.

Results and discussion
The chemical structures of the reactants and products, and an
overview of the chemical reaction system are shown in Fig. 2a.
We selected 7-tetradecene as the alkene source of the hydrophobic moiety and designed a fuel alkene FuelPEG so that theDr. Kazushi Kinbara received aPh.D. degree in organic chemistry from the University of Tokyoin 1996 under the direction ofProfessor Kazuhiko Saigo. From
2001, he joined Professor Takuzo
Aida’s group at School ofEngineering, the University ofTokyo as a lecturer and associate
professor. In 2008, he was promoted to a professor of theInstitute of Multidisciplinary
Researchfor
Advanced
Kazushi Kinbara
Materials, Tohoku University. In
2015, he moved to Graduate School of Bioscience andBiotechnology, Tokyo Institute of Technology. His research interests include (1) bioinpired chemistry and (2) supramolecularchemistry.

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lating “waste” product. We also added FuelPEG to the reaction
mixture as an additional “fuel” after the complete consumption of AmpPEG at 100 min and again observed the transient
formation of AmpPEG (Fig. 2c, red), clearly demonstrating therepeatability of the designed reaction system. Note that theapparent decrease in HydPEG at 100 min (Fig. 2c, green) is dueto the dilution of the reaction mixture upon addition of theaqueous solution of FuelPEG (see the ESI†).
In the previous study by Fletcher and coworkers, theyreported that the ruthenium catalyst was compartmentalized

Fig. 2 (a) Schematic illustration of the designed non-equilibrium
chemical reaction system based on oleﬁn metathesis. (b) Concentration
vs. time plot of the homogeneous reaction mixture using CH2Cl2 as asolvent at 25 °C (480 rpm). (c) Concentration vs. time plot of the biphasic reaction mixture using H2O as a solvent at 50 °C (480 rpm). At
100 min, FuelPEG was added as an additional “fuel”.

resulting amphiphilic metathesis product AmpPEG would
possess the same hydrophobic–hydrophilic balance as Triton
X-114 (Fig. 1) in terms of its constituent atoms. The synthesized 7-tetradecene, FuelPEG, AmpPEG, and HydPEG(vide infra) were unambiguously characterized by NMR andhigh-resolution mass spectrometry (Fig. S1–S13, see the ESI†).
First, we mixed 7-tetradecene, FuelPEG, and Hoveyda–
Grubbs catalyst 2nd generation (HG-II)46,55,56 in CH2Cl2 andmonitored the reaction progress using ultra-performanceliquid chromatography (UPLC) (Fig. S14a, see the ESI†). Notethat all reagents were uniformly dissolved so that the reaction
was performed under homogeneous conditions. As shown in
Fig. 2b, the concentration of AmpPEG (Fig. 2b, red) increased
monotonically in response to the consumption of FuelPEG(Fig. 2b, blue), representing the conventional reaction kinetics.
Next, we used water instead of CH2Cl2 as a solvent to performthe same reaction under biphasic conditions (Fig. S14b, see
the ESI†). Intriguingly, the reaction showed non-equilibrium
kinetics where the concentration of AmpPEG (Fig. 2c, red)reached its maximum at 60 min and then gradually decreased.
In addition, the hydrophilic metathesis product HydPEG(Fig. 2c, green) started to appear after 20 min, following thegeneration of AmpPEG. At 100 min, we observed the complete
consumption of AmpPEG and the exclusive formation of
Hyd
PEG. These results demonstrate that AmpPEG was formed asa transient product, while HydPEG was formed as an accumu-

6444 | Nanoscale, 2024, 16, 6442–6448

Fig. 3 (a and b) Photographs of the mixtures of 7-tetradecene and (a)aqueous FuelPEG (50 mM) and (b) aqueous HydPEG (50 mM) before (left)
and after (right) stirring for 4 h at 25 °C. (c) An intensity-based particle
size distribution proﬁle of aqueous FuelPEG (30 mM) at 20 °C, analyzed
by dynamic light scattering. The mean hydrodynamic diameter of theparticle: 222.2 nm. (d) Transmission electron micrograph of aqueous
Fuel
PEG stained with gadolinium acetate. (e) Surface tension vs. concentration plot of aqueous FuelPEG. The critical aggregation concentration(CAC) was determined from the intersection of the ﬁtted lines([FuelPEG]CAC = 26.7 mM). (f ) Schematic illustration of the mechanism of
the out-of-equilibrium chemical reaction. First, hydrophobic 7-tetradecene and the catalyst are surrounded by FuelPEG in water. Then, thecross-metathesis reaction between 7-tetradecene and FuelPEG occurs at
the interface to form AmpPEG. Subsequently, hydrophilic HydPEG isformed and diﬀuses out of the interface. As a result, the reaction equilibrium is shifted, leading to the complete consumption of AmpPEG. Notethat 7-tetradecene acts as both a reactant and an organic phase.

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Nanoscalewithin the droplet of hydrophobic alkene, and cross-metathesis reactions of hydrophobic and hydrophilic alkene
occurred at the interface with water.36,46 We hypothesized thata similar reaction mechanism also took place in our systemand performed qualitative kinetic simulations. In fact, thesimulation for the biphasic conditions showed that the concentration of AmpPEG increased and then decreased while thatfor the homogeneous conditions did not (Fig. S15, see the
ESI†).
Regarding the reaction mechanism, we also tested theemulsifying properties of FuelPEG and HydPEG by mixing theiraqueous solution with 7-tetradecene. As shown in Fig. 3a,
Fuel
PEG formed turbid dispersions, while HydPEG resulted in
the much-less turbid phase-separated mixture (Fig. 3b). The
diﬀerence in turbidity was also quantified by measuring theoptical density (O.D.) at 600 nm (Fig. S17†). These results
demonstrated that FuelPEG could stabilize the emulsions due
to its amphiphilicity, while HydPEG could not, likely because
Hyd
PEG is too hydrophilic. In fact, using dynamic light scattering (DLS, Fig. 3c), transmission electron microscopy (Fig. 3d),and surface tension measurements (Fig. 3e), we confirmedthat FuelPEG can self-assemble into spherical aggregates inwater while HydPEG cannot (Fig. S19b†). In addition, we performed an optical microscopic observation of the emulsions
formed by 7-tetradecene and FuelPEG in the presence of HG-IIand found that the interior of the emulsions were coloredgreen (Fig. S20†), which is characteristic of the ruthenium
catalyst.57 This result indicates that HG-II was compartmentalized within the droplets of the emulsions as proposed by
Fletcher and coworkers in their earlier study.46 Considering all

Communicationthese results, it seems that the cross-metathesis reaction
between 7-tetradecene and FuelPEG occurred predominantly togenerate AmpPEG in the early stage (Fig. 3f-i). Later, HydPEG
was generated but then diﬀused out of the interface due to its
hydrophilicity, and as a result, HydPEG was excluded from thereaction system, and the reaction equilibrium was further
shifted to lead to the complete consumption of AmpPEG(Fig. 3f-ii).
With the newly developed non-equilibrium chemical reaction system in hand, we then investigated its potential to
exhibit the biologically relevant properties arising from thestructural similarity of AmpPEG to Triton X-114. As model cell
membranes, we prepared giant unilamellar vesicles (GUVs)
formed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) by
the electroformation method (see the ESI†) and added analiquot of the reaction mixture at diﬀerent time points. We
used DilC18(3) to label the GUVs to observe their morphologies
by fluorescence microscopy. Strikingly, we observed a distinct
time-dependent eﬀect; we clearly observed GUVs when we
added the reaction mixture with reaction times of 1 min(Fig. 4a, right) and 120 min (Fig. 4a, left), but we did notobserve any GUVs with a reaction time of 60 min (Fig. 4a,
middle). To quantitatively analyze this phenomenon, we prepared DOPC large unilamellar vesicles (LUVs) encapsulating
5-carboxyfluorescein (5-CF) by the extrusion method and examined the release of 5-CF (see the ESI†). Note that fluorescencequenching of 5-CF occurs when its concentration is high, so
an increase in fluorescence intensity will be observed when it
is released from inside the LUVs.15,58–60 Using authentic
samples of FuelPEG, AmpPEG, and HydPEG, we confirmed that

Fig. 4 (a) Fluorescence micrographs of DiIC18(3)-labeled GUVs ([DOPC] = 600 µM, [DiIC18(3)] = 600 nM) in aqueous glucose (2.0 mM) after the
addition of the reaction mixtures at reaction times of 1 (left), 60 (middle), and 120 min (right) at 25 °C (λex = 520–550 nm, λobsd > 580 nm). (b)Representative ﬂuorescence intensity changes of 5-CF encapsulated in DOPC LUVs ([DOPC] = 100 μM, [5-CF] = 50 mM) in HEPES buﬀer ([HEPES] =20 mM, [NaCl] = 50 mM, pH 7.5) at 20 °C after the addition of the reaction mixtures at diﬀerent reaction times (λex = 490 nm, λem = 520 nm). (c)Overlay of ﬂuorescence intensity changes of 5-CF at 520 nm (left axis, black) and concentration changes of AmpPEG (right axis, red) as a function oftime. Error bars represent standard deviations (n = 3). (d) Overlay of optical density changes of the red blood cell (RBC) suspensions at 540 nm (leftaxis, black) and concentration changes of AmpPEG in the reaction mixture (right axis, red) as a function of time. Error bars represent standard deviations (n = 5). The insets show the photographs of the mixtures of RBC suspensions and reaction mixtures at diﬀerent reaction times.

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CommunicationAmp

PEG eﬀectively ruptured the LUVs, while FuelPEG and
PEG did not (Fig. S24b†). We then measured the fluorescence spectra of 5-CF at diﬀerent reaction time points(Fig. 4b) and plotted their intensity. As shown in Fig. 4c, thefluorescence intensity change showed a good correlation withthe concentration of AmpPEG generated during the reaction.
We also performed DLS measurements of the mixtures andobserved the transient size change from LUVs to smaller aggregates with a diameter of 9.0 nm at a reaction time of 60 min(Fig. S25†), similar to the size of spherical micelles formed byAmp
PEG alone (Fig. S18, S19a and S26†). These results clearly
demonstrated the time-dependent disruption of LUVs causedby the transient generation of AmpPEG, which co-assembled
with DOPC phospholipids to form micelles (Fig. 4a middle,
inset).61,62
To further extend the properties of our non-equilibrium
chemical reaction system, we investigated the possibility oftransient hemolytic activity, the disruption of red blood cells(RBCs), which is highly associated with the maintenance of
homeostasis in the body.63 We added an aliquot of the reaction mixture at diﬀerent time points to the suspensions ofRBCs, incubated the mixture for 30 min, centrifuged, andmeasured the O.D. of the supernatant at 540 nm to quantify
the amount of hemoglobin released by hemolysis (see the
ESI†).64 As shown in Fig. 4d, the O.D. values showed a clear
correlation with the concentration of AmpPEG generated during
the reaction, where high hemolytic activity was observed at thereaction time points of 30 and 60 min. Using authentic
samples of FuelPEG, AmpPEG, and HydPEG, we confirmed thatAmp
PEG is the only hemolytically active compound besides
Fuel
PEG and HydPEG (Fig. S27†). These results clearly demonstrated the first example of transient control of lytic activity bya non-equilibrium reaction system. We also analyzed theabsorption spectra of the mixture and found that the denaturation of hemoglobin did not occur (Fig. S28†), which nicely
corresponds to the membrane-specific activity of Triton X as
mentioned earlier.65

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HydConclusions
We have newly developed a non-equilibrium chemical reaction
system that generates a Triton X-mimetic amphiphile AmpPEG
as a kinetic product. Through a quantitative assay using dyeembedded LUVs, we confirmed that the generation of AmpPEG
enabled time-dependent membrane disruption through interaction with phospholipids. Furthermore, we demonstrated
that the chemical reaction system is applicable to transient
control of the resulting lytic activity. In fact, we successfully
achieved time-controlled hemolysis of RBCs without denaturing hemoglobin. Although further optimization is needed torealize in situ control of biological activity, we believe this work
has opened the door to the development of next-generation
functional supramolecular materials that can precisely andtemporally modulate biological events.

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Nanoscale

Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.

Conﬂicts of interest
The authors declare no competing financial interest.

Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research on Innovative Areas “Molecular Engine” (18H05418,
18H05419, and 23H05418 to K.K.), a Grant-in-Aid for
Challenging Research (Pioneering) (23K17363 to K.K.), a
Grant-in-Aid for Scientific Research (B) (23H02080 to K.S.), a
Grant-in-Aid for Challenging Research (Exploratory) (23K17973
to K.S.), and a Grant-in-Aid for Transformative Research Areas“Molecular Cybernetics” (23H04408 to K. S.). K. S. also thanks
The Salt Science Research Foundation, Iketani Science andTechnology Foundation, The Ube Foundation, and The
Mitsubishi Foundation for their financial support. The
authors thank Suzukakedai Materials Analysis Division, OpenFacility Center, Tokyo Institute of Technology, for NMR spectroscopy, EI mass spectrometry, ESI-TOF mass spectrometry,
TEM observations, and surface tension measurements. In
addition, we acknowledge Yuka Akimoto for her assistancewith TEM. We thank the Open Research Facilities for Life
Science and Technology, Tokyo Institute of Technology, for theuse of a plate reader and an autoclave machine. We also thankProf. Atsushi Maruyama for the use of an ultracentrifuge.

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