Construction of Redox Responsive Vesicles Based on a Supra-Amphiphile for Enzyme Confinement

Article abstract of DOI:10.1002/cjoc.201600772

Redox responsive vesicles were constructed based on self-assembly of a supra-amphiphile built by the host- guest interaction between β-cyclodextrin and a ferrocene derivative. The structure and destruction-reconstruction reversibility of the “smart” vesicles were clearly characterized by electron microscopy and dynamic light scattering. Notably, these vesicles were proved to be capable of controlled release function and bringing protease resistance to the enzyme, showing great biocompatibility and promising applications.

Full text of DOI:10.1002/cjoc.201600772

COMMUNICATION
DOI: 10.1002/cjoc.201600772
Construction of Redox Responsive Vesicles Based on a
Supra-Amphiphile for Enzyme Confinement
‡
‡
Tiezheng Pan, Huixin Zou, Hongcheng Sun, Yao Liu, Shengda Liu, Quan Luo,
Zeyuan Dong, Jiayun Xu, and Junqiu Liu*
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University,
Changchun, Jilin 130012, China
Redox responsive vesicles were constructed based on self-assembly of a supra-amphiphile built by the host-
guest interaction between β-cyclodextrin and a ferrocene derivative. The structure and destruction-reconstruction
reversibility of the “smart” vesicles were clearly characterized by electron microscopy and dynamic light scattering.
Notably, these vesicles were proved to be capable of controlled release function and bringing protease resistance to
the enzyme, showing great biocompatibility and promising applications.
Keywords redox responsive, host-guest systems, vesicles, supra-amphiphile, glutathione S-transferase
[
9]
Introduction
modulated by redox stimulus. In reduced state, ferro-
cene derivatives form stable 1∶1 complexes with β-CD
in water by penetrating the internal hydrophobic cavity
of β-CD. However, the oxidation of ferrocene leads to a
significant decrease of the host-guest interaction be-
tween β-CD and the ferrocenium cation, dramatically
diminishing the stability of the complex. In this regard,
the attachment of ferrocene to β-CD should offer great
possibilities for the construction of transformable struc-
tures and redox switchable vesicles.
Most of the biomacromolecules in cells are delicate-
ly confined by membranes for the compartmentalization
[
1]
of biochemical reactions. Especially, enzymes as effi-
cient biocatalysts are well organized for parallel and
multistep reactions, obviating the need for functional
group protection of substrates and functioning in intri-
[2]
cate order. In practical applications, many immobili-
zation techniques have endowed the enzymes with im-
[3]
proved stability, activity and selectivity. Therefore, the
confinement of enzymes is of great significance for both
academia and industry. Along with the development of
supramolecular chemistry and material science, tre-
mendous stimulus-responsive materials emerged to
bring abundant “smart” materials that are available for
In this work, we report the construction of redox re-
sponsive vesicles built by the host-guest interaction be-
tween β-CD and a ferrocene derivative Fc-1. The as-
sembly and dissociation of the vesicles could be con-
trolled by the redox states of the ferrocene head of Fc-1.
Furthermore, we confined the active enzyme glutathione
S-transferase (GST) within the vesicles, and its con-
trolled release was demonstrated by monitoring the GST
activity shift in the solution. GSTs play an essential role
in the metabolic detoxification processes and catalyze
the exogenous substances into more hydrophilic and
less toxic polypeptide derivatives using glutathione
GSH) as cofactor. Due to the fact that cell detoxifi-
cation requires oxidative stress provided by xenobi-
otic-metabolizing enzymes such as cytochrome P450,
these “smart” redox responsive vesicles with loaded
GST together may present a potential antidote against
electrophilic aromatic compounds with selectivity at
oxidative environment.
[
4]
enzyme confinement. Among them, vesicles with
switchable morphologies and controlled release func-
tions have been widely studied for cell mimicking and
[5]
drug delivery.
In recent years, many amphipathic molecules, espe-
cially supra-amphiphiles have been used to construct
[6]
vesicles. Particularly, the biocompatible and degrada-
ble properties of cyclodextrin (CD) make CD-based
vesicles excellent candidates for the confinement and
[
10]
(
[11]
[
7]
transport of biomacromolecules. Also, CDs are known
to form supra-amphiphilic inclusion complex with a
series of guest molecules such as adamantane, azoben-
zene and ferrocene derivatives. Especially, the binding
capacity of ferrocene derivatives to β-CD could be
[
8]
*
E-mail: junqiuliu@jlu.edu.cn; Tel.: 0086-0431-85168452
Received November 8, 2016; accepted December 27, 2016; published online XXXX, 2017.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201600772 or from the author.
These authors contributed equally to this work.
‡
In Memory of Professor Enze Min.
Chin. J. Chem. 2017, XX, 1—5
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Pan et al.
COMMUNICATION
Scheme 1 Schematic representation of the designed redox responsive vesicles and the controlled release of GST
Experimental
Figure 1A, the SEM image confirmed the existence of
the spherical morphology of the vesicles with a diameter
ranging from 250 to 450 nm. The concentrations of
β-CD and the guest molecule are critical to the mor-
The ferrocene-based guest molecule Fc-1 which
contained three hydrophobic tails was synthesized (Fig-
ure S1), and its structure was confirmed by NMR analy-
sis (Figures S2 and S3). Then the assembly process was
carried out by dissolving β-CD and the Fc-1 respective-
4
1
phology, as 1×10 mol•L was proven to be the opti-
mal concentration for the assembly process (Figure
S4A). Also, the guest molecule Fc-1 was individually
characterized by SEM under the same condition, and no
regular assemblies were observed (Figure S4B), demon-
strating that the vesicles were constructed through the
host-guest interaction between β-CD and Fc-1. To fur-
ther explore the detailed structure of the spherical as-
sembly, transmission electron microscopy (TEM) ob-
servation was conducted. Rather than a solid micelle,
Figure 1B evidently showed a hollow structural vesicle
with an apparent contrast between the periphery and the
internal position. According to the picture, the thickness
of the vesicle was determined to be 6 nm, which was
quite close to the thickness of the supra-amphiphile bi-
layer (calculated result from GaussView and PyMOL
softwares). These electron microscope images together
proved that the supramolecular vesicles were success-
fully constructed through the host-guest interactions
between β-CD and Fc-1.
1
ly in DMF to the same concentration (20 mmol•L ),
and they were slowly mixed together at a ratio of 1∶1
under ultrasonic for 20 min. The complex was then di-
luted to a final concentration of 0.1 mmol•L1 under
ultrasound for 30 min.
3
The dissociation of the vesicles required FeCl at
room temperature. To rule out the possibility that the
cation might affect the assembly morphology, excessive
ZnSO (1 mmol•L ) was also added into the solution of
4
vesicles for SEM characterization (Figure S4).
1
In the enzyme encapsulation experiments, the GST
variant was over-expressed in Escherichia coli and puri-
fied using a Glutathione Sepharose 4B column for the
following experiments (Figure S5). Then the enzyme
was encapsulated into the vesicles in the assembly pro-
cess of β-CD/Fc-1 complex, and the concentration of
the enzyme was determined to be 10 mol•L by
staining of coomassie brilliant blue, and the redundant
free GST was removed by dialysis. The encapsulation
efficiency was determined to be 11% by detecting the
protein concentration using Bradford method. Next, the
6
1
3
＋
release of GST in the absence and presence of Fe was
monitored respectively by an activity assay:
The reaction rate is proportional to the enzyme con-
centration under the experimental conditions. Also, the
product of the reaction has a maximum absorption peak
at 340 nm, which is different from GSH or 1-chloro-
Figure 1 SEM and TEM images of the vesicles. (A) SEM
characterization of β-CD and Fc-1 assembled at 1×10 mol•L .
(B) TEM characterization of a single vesicle.

4
1
2
,4-dinitrobenzene.
Because of the redox-responsive properties of
β-CD/Fc-1 complex, the oxidation mediated dissocia-
tion of the assembly was also observed. By adding the
Results and Discussion
The morphology of the assemblies was characterized
by scanning electron microscopy (SEM). As shown in
3
＋
oxidizing agent Fe , the ferrocene head of Fc-1 would
2
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Construction of Redox Responsive Vesicles
be oxidized and escape from the hydrophobic cavity of
β-CD, leading to the dissociation of the vesicles. This
transformation process was captured by SEM (Figures
finement and controlled release experiments were con-
ducted by taking advantage of the redox-responsive
feature of the vesicles. The connection between oxida-
tive stress and the working circumstance of GST makes
it perfect cargo to study the catalytic behaviours during
the confinement and release processes.
2
A and 2B). In addition, non-oxidizing transition metal
2
＋
Zn was added into the assembly system under the
same condition to rule out the possibility that the cation
might affect the morphology by changing the ionic
strength. The SEM image showed that the vesicle struc-
ture remained unchanged (Figure S4C), proving that the
Evident activity distinction was observed between
the confined and released groups (Figure 3A). To be
more specific, the release ratio of GST in the blank
3
＋
3＋
dissociation was caused by the oxidative ability of Fe .
Moreover, the vesicle morphology exhibited poten-
tial reversibility feature according to the redox agents in
the system. An important biogenic cofactor, GSH is
widely used as a reducing agent in organisms and
man-made systems. Therefore, we monitored the kinet-
ics process of the formation and dissociation behaviour
group without Fe was determined to be 2.1% after 10
min and 10.9% after 50 min, proving that the enzyme
confinement was relatively stable. On the contrary, the
release ratio dramatically increased to 25.6% after 10
min and 95.8% after 50 min with the addition of the
3
＋
Fe (Figures 3B and S8). As the vesicles were contin-
uously damaged by excessive oxidizing agent, GST was
completely released after 1 h, which was consistent with
the dissociation kinetics of the vesicles demonstrated by
DLS.
3
＋
of the assembly by alternately adding GSH and Fe .
Dynamic light scattering (DLS) data indicated that the
average diameter of the assembly kept decreasing upon
3
＋
the addition of Fe , eventually large scale assembly
completely dissociated. However, the ferrocene head of
Fc-1 could be reduced by GSH to regain the host-guest
recognition with β-CD, and the average diameter gradu-
ally increased to a maximal value which was almost
consistent with that of the initial assembly (Figure S6).
Remarkably, the destruction-reconstruction cycle was
repeatable for several turnovers without dramatic atten-
uation (Figure 2C). These results demonstrated that the
vesicles were accurately redox-controlled with complete
reversibility.
Figure 3 (A) The reaction rates of the blank control without
GST (a), the confined GST (b), the released GST (c) and the free
state GST in aqueous solution (d) by monitoring the reaction
product at λ＝340 nm. (B) The GST release percentage of the
GST-containing vesicles in the absence (a) and in the presence of
Figure 2
SEM images of the morphologies in the re-
duced/oxidized states and the destruction-reconstruction process
monitored by DLS: (A) Integrated vesicles in the reduced state
and (B) dissociated vesicles in the oxidized state. (C) The revers-
3
+
excessive Fe (b).
3
+
ibility curve of vesicle diameters conducted by adding Fe /GSH.
As expected, the release could be finely controlled
by the concentration of the oxidizing agent. By increas-
3
＋
The vesicles with hollow internal structure are per-
fect nanocontainers for molecular embedding, material
transport and drug delivery. Therefore, the enzyme con-
ing the Fe in the beginning, more GST molecules
were released after 1 hour’s incubation (Figure 4A).
Moreover, the S-shaped curve not only afforded an IC50
Chin. J. Chem. 2017, XX, 1—5
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Pan et al.
COMMUNICATION
1
3＋
of 0.08 mmol•L Fe for GST release, but also indi-
cated a mechanism that the disassembly rate was first
accelerated then slowed down. So in the presence of low
3
＋
concentration of Fe , the minority disassembled su-
pra-amphiphiles did not lead to immediate dissolution
3
＋
of the vesicles. However, with more and more Fe
added into the system, the integrity of the vesicles was
dramatically compromised with increasing velocity.
Figure 5 SDS-PAGE of the trypsin digestion of GST. Lane 1:
the GST in the absence of trypsin. Lane 2: the confined GST
incubated with trypsin. Lane 3: the free state GST incubated with
trypsin.
Conclusions
In conclusion, we have conducted the confinement
of GST using well-defined vesicles formed by the con-
struction of a supra-amphiphile through the host-guest
interaction between β-CD and the ferrocene derivative
Fc-1. Due to the redox features of the ferrocene head of
3
＋
Fc-1, adding oxidizing agent Fe resulted in the disso-
ciation of the vesicles. Also, this process was reversible
upon the addition of GSH that could reduce the oxidized
ferrocene head to promote the re-assembly process.
Hence, the confined GST was able to be released with
precise control by the incubation time and concentration
of the oxidizing agent. Moreover, the stability of the
enzyme inside the vesicles was dramatically enhanced
in the presence of protease. These “smart” vesicles were
proved to be promising nanocontainers for enzymes,
and the confinement strategy may have potential appli-
cations in biomacromolecule transport and drug deliv-
ery.
Figure 4 (A) The GST controlled release percentage by differ-
3
+
ent Fe concentration. (B) The reaction rates of blank control
without GST (a), trypsin treated free GST (b), trypsin treated
confined GST (c) and free GST without trypsin (d).
Furthermore, the confined GST in vesicles and free
state GST were both treated with trypsin, an efficient
protease which could induce the enzyme to proteolysis.
The result showed that the free GST clearly lost the cat-
alytic ability, while the confined GST still remained the
activity (Figure 4B). In addition, the trypsin-treated
GSTs were characterized by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Figure 5 showed that the
free GST (Lane 3) was digested into oligopeptides,
while the confined GST (Lane 2) remained constant
with the untreated enzyme (Lane 1) in molecular weight.
These results proved that the vesicles were able to
shield the enzyme from proteolysis. In this way, the en-
zyme confinement and controlled release by the vesicles
were demonstrated through the GST activity assays,
presenting redox responsive and protease-stable nano-
containers with good biocompatibility.
Acknowledgement
This work was supported by the National Natural
Science Foundation of China ((Nos. 21420102007,
2
1234004, 21574056, 91527302), the Chang Jiang
Scholars Program of China, and Project 2016014 Sup-
ported by Graduate Innovation Fund of Jilin University.
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