Construction of a smart temperature-responsive GPx mimic based on the self-assembly of supra-amphiphiles

Article abstract of DOI:10.1039/c5sm02074c

Glutathione peroxidase (GPx) is a major defense against hydroperoxides as a kind of seleno-enzyme that protects cells from oxidative damage. A supramolecular vesicle with controllable GPx activity and morphology has been successfully constructed by the self-assembly of supra-amphiphiles formed by host-guest recognition between cyclodextrin and adamantane derivatives. By introducing thermosensitive poly(N-isopropylacrylamide) (PNIPAM) scaffolds and the catalytic moiety selenium into adamantane and cyclodextrin, respectively, the complex of catalysis-functionalized cyclodextrin with thermosensitivity-functionalized adamantane directed the formation of a supramolecular vesicle which acted as a GPx mimic at 37 °C. The self-assembled nanoenzyme exhibited an obvious temperature responsive characteristic and high GPx-like catalytic activity promoting the reduction of hydrogen peroxide (H₂O₂) with glutathione (GSH) as the reducing substrate at 37 °C. However, the vesicle disassembled when the temperature decreased to 25 °C due to the transition of PNIPAM between the coil and the globule. Interestingly, the catalytic activity changed along with the transformation of morphologies. The vesicle structure self-assembled at 37 °C provided the favorable microenvironment for the enzymatic reaction, hence we successfully developed a temperature-responsive nanoenzyme model. Moreover, the catalytic activity of the thermosensitive GPx mimic exhibited excellent reversibility and typical saturation kinetics behaviour similar to a natural enzyme catalyst. It is assumed that the proposed GPx model not only has remarkable advantages such as easy functionalization and facile preparation but also provided a new way to develop intelligent responsive materials.

Full text of DOI:10.1039/c5sm02074c

Soft Matter
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Construction of a smart temperature-responsive
GPx mimic based on the self-assembly of supra-
amphiphiles†
Cite this: Soft Matter, 2016,
12, 1192
Huixin Zou, Hongcheng Sun, Liang Wang, Linlu Zhao, Jiaxi Li, Zeyuan Dong,
Quan Luo, Jiayun Xu and Junqiu Liu*
Glutathione peroxidase (GPx) is a major defense against hydroperoxides as a kind of seleno-enzyme
that protects cells from oxidative damage. A supramolecular vesicle with controllable GPx activity and
morphology has been successfully constructed by the self-assembly of supra-amphiphiles formed by
host–guest recognition between cyclodextrin and adamantane derivatives. By introducing thermosensitive
poly(N-isopropylacrylamide) (PNIPAM) scaffolds and the catalytic moiety selenium into adamantane and
cyclodextrin, respectively, the complex of catalysis-functionalized cyclodextrin with thermosensitivity-
functionalized adamantane directed the formation of a supramolecular vesicle which acted as a GPx
mimic at 37 1C. The self-assembled nanoenzyme exhibited an obvious temperature responsive
characteristic and high GPx-like catalytic activity promoting the reduction of hydrogen peroxide (H2O2)
with glutathione (GSH) as the reducing substrate at 37 1C. However, the vesicle disassembled when the
temperature decreased to 25 1C due to the transition of PNIPAM between the coil and the globule.
Interestingly, the catalytic activity changed along with the transformation of morphologies. The vesicle
structure self-assembled at 37 1C provided the favorable microenvironment for the enzymatic reaction,
hence we successfully developed a temperature-responsive nanoenzyme model. Moreover, the catalytic
activity of the thermosensitive GPx mimic exhibited excellent reversibility and typical saturation kinetics
behaviour similar to a natural enzyme catalyst. It is assumed that the proposed GPx model not only has
remarkable advantages such as easy functionalization and facile preparation but also provided a new
way to develop intelligent responsive materials.
Received 19th August 2015,
Accepted 17th November 2015
DOI: 10.1039/c5sm02074c
www.rsc.org/softmatter
(GSH) as a reducing substrate.4,5 It is apparent that mimicking
the excellent antioxidation performance of natural GPx without
Introduction
Enzymes, serving as a class of highly specific and efficient the intrinsic disadvantages is of vital importance. On the other
biocatalysts in nature, enhance the rates of reactions between hand, building reliable artificial GPx models in a smart manner
the metabolites in vivo. The design of artificial enzymes repre- remains challenging and significant. In chemists’ hands, remark-
sents one of the increasingly significant topics in the research able accomplishments have been obtained by introducing selenium
of biomaterials. Reactive oxygen species (ROS), which are catalytic centers into the substrate-binding scaffolds, including
produced by the cellular metabolic process, play an essential chemical and biological modifications. In particular, in the case
role in cell signaling.1 They are friendly to the living organisms of some chemical methods, seleno-dendrimers6 and selenium-
in general, nevertheless, the release of surplus ROS may also or tellurium-containing interactional small organic compounds7,8
result in oxidative damage and eventually some diseases, such as are typical examples. In recent years, our group has focused
neuronal apoptosis, cardiovascular diseases and inflammatory on the construction of highly efficient selenium-containing
processes.2,3 Glutathione peroxidase (GPx) (EC 1.11.1.9) is a enzymes based on the understanding of the structure of a
crucial selenoenzyme involved in scavenging ROS by catalyzing native GPx by the incorporation of catalytic centers with existing
the reduction of hydroperoxides (ROOHs) using glutathione or artificially generated scaffolds.9–13
Achievements in supramolecular chemistry provide strong
incentives to construct artificial enzymes. However, establishing
an activity-controllable nanoenzyme system has become an over-
State Key Laboratory of Supramolecular Structure and Materials, College of
Chemistry, Jilin University, Changchun 130012, People’s Republic of China.
arching goal in the design of smart artificial enzymes. To meet
E-mail: junqiuliu@jlu.edu.cn; Fax: +86-431-85193421
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sm02074c the challenge, stimulus-responsive architectures with chemical
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Scheme 1 Schematic representation of the self-assembly process based on host–guest recognition and the temperature responsive behaviour of the
GPx mimic.
triggers14,15 have been designed to provide the capability to water solution, which resulted in a relatively low catalytic efficiency.
control the catalytic activity of GPx mimics. In the meanwhile, Whereas with the chains becoming coiled at 37 1C, the polymer
the intelligent analogues of enzymes in response to a variety of turned hydrophobic, thus leading to the formation of a supra-
factors have attracted growing attention, such as thermal,16 amphiphilic molecule. It further self-assembled into vesicles and
photoresponsive,17 redox18 and even mechanical forces.19 Among the selenium catalytic centers were aggregated and exposed on the
these stimulus-responsive artificial enzymes, poly(N-isopropyl- surface. The supra-amphiphilic vesicles might thus offer an out-
acrylamide) (PNIPAM), acting as a smart material, has been standing model for fabricating a GPx mimic with high catalytic
widely investigated due to its thermal responsive properties. activity by centralizing the catalytic centers together (Scheme 1).
Owing to its transition between the coil and the globule upon Furthermore, the catalytic activity of this smart artificial GPx
temperature changes, PNIPAM presents a dramatic phase transi- mimic can be reversibly modulated by temperature.
tion in water. The temperature related to conformational changes
is defined as the lower critical solution temperature (LCST).20–22
When the temperature is below the LCST, the PNIPAM block is
Materials and methods
Materials
hydrophilic whereas hydrophobic above the LCST.23 Compared
to other thermal responsive nanomaterials, the unique change in
the physical properties of PNIPAM has received much attention
from researchers in fabricating thermal responsive assemblies.
hexane with a proportion of 3 : 7. Tris(2-dimethylaminoethyl)-
To the best of our knowledge, the self-assembly by host–guest
N-Isopropylacrylamide (NIPAM) was purchased from Sigma-
Aldrich and recrystallized from the mixture of toluene and
amine (Me6TREN), glutathione reductase (GR), glutathione
(GSH) and b-nicotinamide adenine dinucleotide phosphate
recognition has been investigated as an effective tool to construct
functional nanostructures in aqueous media with broad applica-
tion prospects.24–28 In addition, vesicles, fibers or nanotubes
(NADPH) were purchased from Sigma-Aldrich and used directly.
Sodium borohydride (NaBH4) and H2O2 were purchased from
constructed by the assembly of amphiphiles are not only common
Sinopharm Chemical Reagent Co. Ltd and were used without
building blocks in living systems but also fundamental compo-
further purification. Cuprous bromide (CuBr) was synthesized from
nents of artificial biomimetic systems, molecular imprinting
systems, microreactors, and especially drug delivery systems.13,29
cupric bromide (CuBr2) and sodium sulfite (Na2SO3). Selenium
powder, 4-toluene sulfonyl chloride (PTSC) and b-cyclodextrin
were purchased from Aladdin Industrial Corporation, among
which b-cyclodextrin was recrystallized three times before use.
Milli Q water was used in the assembly process and other
Herein, we designed a supra-amphiphilic thermosensitive vesicle-
like structure by the self-assembly of host–guest interaction
between cyclodextrin (CD) and adamantine (Ad) derivatives to
construct a novel artificial GPx mimic with controllable catalytic
solvents were of analytical grade.
activity. Specifically, Ad-headed PNIPAM (Ad-PNIAM, Scheme 1,
1b) served as the guest molecule while cyclodextrin-based selenic
acid (CD-Se, Scheme 1, 2a) acted as the host in this complex.
Instruments and measurements
At 25 1C, the host–guest complexation events occurred in A Bruker Advance 500 (500 MHz) 1H NMR spectrometer was
aqueous solution and the PNIPAM chains had high-solubility employed to characterize the structures of the compounds.
below the LCST, meanwhile the complex became hydrophilic Molecular weights were determined by MALDI-TOF, ESI-MS
and the selenium catalytic centers were distributed randomly in spectrometric analyses and GPC. Scanning electron microscopy
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(SEM) observations were carried out on a JEOL FESEM 6700F
scanning electron microscope where the primary electron
energy is 3.00 kV. Transmission electron microscopy (TEM)
observations were carried out using a JEM-2100F scanning electron
microscope. The UV-vis spectroscopy spectrum was supervised
using a Shimadzu 2450 UV-VIS-NIR spectrophotometer. And a
Malvern Zetasizer NanoSeries instrument was used to monitor
the size of the vesicle.
Synthesis of the initiator compound 1
Amantadine (1.0 g, 6.6 mmol) and desiccative trimethylamine
(1.8 mL, 13.2 mmol) were dissolved in 20 mL of anhydrous
dichloromethane in a 100 mL round flask. 2-Bromoisobutyryl
bromide (0.8 mL, 6.6 mmol) was dissolved in 10 mL of anhydrous
dichloromethane and added dropwise to the aforesaid round flask
in an ice-bath and stirred for 12 h. The solution was then extracted
from water and dichloromethane. The organic phase was collected
and the resulting product was purified by column chromatography
(silica gel, dichloromethane). Yield: 85%. 1H NMR (500 MHz,
CDCl3) d 2.15–2.10 (3 H, –CH–), 2.05 (6 H, –CH2–), 1.74–1.70
(6 H, –CH2–), 1.95 (6 H, –CH3).
Scheme 2 Synthetic route of (a) Ad-PNIPAM and (b) CD-Se.
spectrophotometer at different temperatures from 25 1C to 45 1C.
The LCST of PNIPAM-CD was found to be 32.4 1C.
Synthesis of Ad-PNIPAM (2)
Preparation of GPx mimics
Ad-PNIPAM was synthesized according to the polymerization
procedure reported by Masci et al.30 NIPAM (848.0 mg, 7.5 mmol),
Me6TREN (23.0 mg, 0.1 mmol), and DMF (5.0 mL) were intro-
duced into a Schlenk tube equipped with a magnetic bar and an
oil bath followed by three freeze–pump–thaw cycles. And then,
CuBr (14.4 mg, 0.1 mmol) was added under nitrogen followed by
two freeze–pump–thaw cycles. Finally, initiator compound 1
(30.0 mg, 0.1 mmol) was deoxygenized and added to the Schlenk
tube via a syringe to start the polymerization. The mixture was
stirred for 48 h at 60 1C. The mixture was then exposed to air to
terminate the polymerization and the solution was dialyzed
against water for 3 days. The resulting solution was freeze-
b-CD, CD-Se and Ad-PNIPAM were dissolved in DMF at a
concentration of 20 mM in three Eppendorf tubes, mixed them
together at a ratio of 4 : 1 : 5, and sonicated for 20 min to get the
complex in high concentration (10 mM) at 25 1C (below the
LCST of Ad-PNIPAM). The stock solution was diluted with Milli-Q
water to a final concentration of 0.1 mM and the temperature
was raised to 37 1C which was above the LCST of NIPAM. The
above solution was sonicated for 20 min to prepare the GPx
mimics and the opalescence appeared.
LCST determination of the assembled complex
1
dried using a lyophilizer (Scheme 2a). H NMR analysis of the
The same method as that of Ad-PNIPAM was carried out to
detect the optical transmittance of the assembled complex
(PBS, 0.1 mM, pH = 7) solution at different temperatures. And
the LCST of the assembled complex was 33.2 1C. Compared
with the GPx mimic, the LCST of the complex which was
comprised of Ad-PNIPAM and b-CD was found to be 33.4 1C.
polymer revealed a ratio of NIPAM : Ad of 65 : 1 (Fig. S5, ESI†).
The GPx mimic showed high activity and was provided with
excellent temperature-responsive effects at this ratio.
Synthesis of CD-Se (4)
6,60-Selenium bridged bis-6-deoxy-b-cyclodextrin (6-CD-SeSe-CD, 3)
was prepared as described previously except that tellurium powder
was replaced by selenium powder.31 6-CD-SeSe-CD (100.0 mg,
0.04 mmol) was dissolved in Milli Q water (20 mL), and then a
large excess amount (30%, 200 mL) of H2O2 was added in an ice-
bath and stirred overnight. The resulting product was purified
by Sephadex G-15 column chromatography with Milli Q water
as the eluent (Scheme 2b). A white solid product 4 was obtained
by freeze-drying (41.6 mg, 0.03 mmol, 80%).
Determination of GPx catalytic activity
GPx activity was determined using H2O2 and GSH as substrates
on the basis of the previous method reported by Wilson33 (see
the following chemical equation).
Enzyme
ƒƒƒƒ!
2GSH þ H2O2
GSSG þ H O
2
Glutathione reductase
GSSG þ NADPH þ Hþ
2GSH þ NADPþ
ƒƒƒƒƒƒƒƒƒƒ!
LCST determination of Ad-PNIPAM
The reaction was carried out at 37 1C in a 500 mL quartz
The determination of LCST of Ad-PNIPAM was carried out cuvette of phosphate buffer (pH = 7.0, 50 mM) containing 1 mM
according to the previously reported recipes.32 We measured GSH, 1 mM EDTA, 1 unit of glutathione reductase and 0.01 mM
the optical transmittance of the Ad-PNIPAM solution (0.1 mM, GPx mimic (calculated based on the concentration of selenium).
pH = 7, PBS) at 600 nm using a Shimadzu 2450 UV-VIS-NIR In this coupled reductase assay system, heat is preserved for
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3 min, and then 50 mL of NADPH (0.25 mM) was added.
The mixture was preincubated for 3 minutes and the reaction
was then initiated by the addition of 50 mL of H2O2 (0.50 mM).
Since the UV spectrum of NADPH exhibits an intense absorption
peak at 340 nm, the initial rates for the reduction of H2O2 by GSH
would be expected to be determined by monitoring the absorption
decrease of NADPH at 340 nm using a Shimadzu 2450 UV-VIS-NIR
spectrophotometer. Appropriate control of the non-enzymatic
reaction was assayed and subtracted from the catalyzed reaction.
Fig. 2 (A) SEM image and (B) TEM image of the GPx mimic at 37 1C.
Results and discussion
LCST behaviour
guest molecules inside to form a 1 : 1 complex.36–41 Involving the
host–guest complex formation between CD and an Ad-headed
PNIPAM guest molecule, our process builds a novel supramolecular
amphiphilic assembly. The CD moiety acted as the hydrophilic
part while PNIPAM shrinked and served as the hydrophobic
portion at 37 1C (above the LCST). This amphipathic molecule
may focus intense effort on the further self-assembly. The
morphology of the GPx mimic was characterized by scanning
electron microscopy (SEM). As shown in Fig. 2A, the SEM image
confirmed the existence of the spherical morphology of the GPx
mimic at 37 1C with a diameter ranging from 200 to 400 nm. In
contrast, the vesicular morphology could hardly be observed at
25 1C (Fig. S8, ESI†). It could be easily assumed that host–guest
recognition has taken place and the complex was totally hydro-
philic under these conditions at 25 1C, which restricted the
further self-assembly.
To further explore the detailed structure of the spherical
assembly, TEM observation was carried out. Fig. 2B demon-
strates a striking contrast between the periphery and the inner
part of the spherical nanoparticles, which is a typical charac-
teristic for hollow vesicles. And the wall thickness of the vesicle
is about 37 nm. In comparison, TEM observation was also
carried out on the guest molecule (Ad-PNIPAM) under the same
conditions (Fig. S9, ESI†). It turned out that the morphology of
the guest molecule was a solid micelle and the diameter was
about 50 nm at 37 1C, which was different from the assemblies.
All these results proved that we have successfully developed a
vesicle-like GPx mimic. Moreover, we employed X-ray diffrac-
tion (XRD) (Fig. 3) to further offer detailed information about
the layered structure of the vesicle. The thickness of each layer
was calculated to be 1.45 nm according to the Bragg equation
and the wall of the vesicle was composed of about 26 layers. The
result of XRD is another powerful evidence for the formation of
regular vesicle-like architectures and the preparation of the GPx
mimic. The vesicle-like GPx mimic confirmed by SEM, TEM,
and XRD was accumulated by many layers, therefore forming a
well-ordered superstructure as shown in Scheme 1. The hydro-
philic CD faced outside whereas the hydrophobic polymer
chain shrinked interiorly.
In our study, on account of the temperature responsive behaviour of
the guest molecule, when the temperature exceeded the LCST, the
soluble PNIPAM transformed into an insoluble polymer that served
as the hydrophobic block, while CD performed the hydrophilic part.
The complex showed excellent temperature-responsive behaviour
and provided the platform to construct universal self-assembled
materials.34,35 Hence, it is a necessity to investigate the thermal-
responsive properties of the polymer Ad-PNIPAM. The control-
lable structural change of PNIPAM can be realized by modulating
the temperature of the complex. Fig. 1 shows the optical trans-
mittance of the guest molecule Ad-PNIPAM (curve a) and the
assembly (curve b) at different temperatures. The LCST of
Ad-PNIPAM and the complex was 32.4 1C and 33.2 1C, respectively,
which suggested that the introduction of hydrophilic CD and
CD-Se with appropriate proportion as the nestable container for
Ad-PNIPAM through host–guest interaction mildly changes the
LCST. In all cases, it is clear that the polymer with temperature-
responsive feature is able to control its own hydrophilic–hydrophobic
properties by temperature.
Construction and characterization of the nanoenzyme
CD is a typical member of host family, which has a cavity and
exhibits brilliant host–guest properties including encapsulating
The temperature dependence of the hydrodynamic diameters
of the assemblies and the guest molecule was further character-
ized by dynamic light scattering (DLS). As shown in Fig. 4, the
current soluble Ad-PNIPAM adopted an extended conformation
and exhibited a diameter of about 10.1 (ꢀ1.0) nm at 25 1C.
Fig. 1 Optical transmittance at 600 nm obtained for solutions (pH = 7.0,
50 mM PBS) of (A) Ad-PNIPAM, (B) GPx mimic and (C) complex of Ad-PNIPAM
and b-CD at a concentration of 0.1 mM.
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Table 1 GPx activities of one catalytic center on supramolecular nanoen-
zymes for the reduction of H2O2 by GSH at pH 7.0 and various
temperatures
Number
Catalyst
Temperature (1C)
n0 (mM minꢁ1
a
)
1
2
3
4
5
6
7
8
GPx mimic
GPx mimic
GPx mimic
GPx mimic
GPx mimic
GPx mimic
GPx mimic
GPx mimic
GPx mimic
GPx mimic
PhSeSePhb
PhSeSePhb
25.0
28.0
30.0
32.0
34.0
35.0
37.0
39.0
41.0
43.0
25.0
37.0
1.3 ꢀ 0.08
3.0 ꢀ 0.2
4.9 ꢀ 0.3
5.7 ꢀ 0.3
6.9 ꢀ 0.3
9.7 ꢀ 0.4
19.9 ꢀ 0.6
18.6 ꢀ 0.5
17.3 ꢀ 0.5
17.2 ꢀ 0.5
0.03 ꢀ 0.001
0.2 ꢀ 0.008
9
10
11
12
a
The GPx activity was corrected for spontaneous reaction in the
presence of the enzyme (2 mM) and assuming one molecule catalytic
center (selenium moiety) as one active site of the enzyme. The thermo-
sensitive nanoenzyme was constructed by 1a, 2a and 1b (Scheme 1).
Fig. 3 XRD scan of the vesicle-like assemblies.
b
The concentration of catalyst PhSeSePh was 10 mM. All the results are
means of at least three repeats.
regulating the conformation of the thermosensitive polymer
PNIPAM that linked to the guest molecule, the construction of
the temperature-responsive GPx mimic was realized. To inves-
tigate the temperature responsive properties of the GPx mimic,
the activity of the nanoenzyme was determined with the tem-
perature ranging from 25 to 43 1C, and the activity values are
shown in Table 1 and by plotting the catalytic activity against
temperature, an optimum temperature curve was obtained
(Fig. 5B). The corresponding blank experiments without mimic
enzyme were also carried out to deduct the background. As the
temperature increases to LCST, the catalytic activity increased
slowly with low magnitude. However, it is noted that with
increasing temperature further up to 37 1C, the activity values
had a sharp promotion due to the formation of stable assem-
blies and the effect of the vesicle structure that provided the
Fig. 4 The hydrodynamic diameters of Ad-PNIPAM (b and c) and GPx
mimic (a and d) determined using a dynamic light scattering instrument,
and the concentration of the sample was 0.1 mM in water. From (a) to (d),
the temperature was 25 1C, 37 1C, 25 1C, and 37 1C, respectively.
When the temperature climbed to 37 1C (higher than the LCST),
the chain aggregated and then self-assembled into micelles. As
a result, the hydrodynamic diameters became 68.1(ꢀ3.5) nm
(curve c). Nevertheless, with the addition of the host molecule
CD, only host–guest recognition occurred at 25 1C (curve a,
8.7 (ꢀ0.5) nm) and an increase in temperature gradually led to
the conformational change of PNIPAM, which further resulted
in the formation of the supra-amphiphilic assemblies. The phenom-
ena above were mainly reflected in the increase of hydrodynamic
diameters (curve d, 342.0 (ꢀ18.0) nm) at 37 1C which was
approaching to what we have observed in SEM images. All these
results demonstrated that the temperature controlled amphi-
pathic GPx mimic fabricated by supramolecular interactions was
successfully constructed.
Catalytic behaviour of the GPx mimic
We modified selenium onto the host molecule CD by chemical
modification to simulate the function of GPx in our well-designed
architecture. The activity of the GPx mimic for the reduction of
H2O2 by GSH was measured according to a coupled reductase
assay system. The absorbance at 340 nm was registered to
calculate the reaction rate on the basis of the decreased
absorption value of NADPH. In our nanoenzyme model, by
Fig. 5 Plots of the catalytic rate of (A) complex of Ad-PNIPAM, b-CD and
Se-CD and (B) the GPx mimic vs. temperature during the catalytic reduction
of H2O2 (0.5 mM) by GSH (1 mM) with catalytic center 2.00 mM. The range of
temperature is from 25 to 43 1C.
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favorable environment for catalytic reaction. Subsequently, the
catalytic activity decreased slightly when the temperature
further increased above 37 1C, which demonstrated that 37 1C
was found to be the optimal temperature for our GPx mimic.
The real-time catalytic plots of absorbance versus time during
the catalytic reduction are displayed in the ESI† in Fig. S10. On
the one hand, increasing the temperature results in the con-
formational change of PNIPAM and the occurrence of the
amphiphilic self-assembly, in which case the assemblies were
proved to be vesicle structures and the active site Se was
gathered and exposed to the substrates, efficiently facilitating
the binding between the substrates and the catalytic centers.
On the other hand, owing to the unique structure of the supramo-
lecular amphiphilic nanoenzyme, the catalytic capability in each
catalytic center presents a remarkable promotion. As expected, the
vesicle-like GPx mimic exhibited a relatively high catalytic activity
(19.9 ꢀ 0.6 mM minꢁ1) at 37 1C. In contrast, at 25 1C the complex
showed rather low catalytic activity (1.3 ꢀ 0.08 mM minꢁ1) (Table 1).
Moreover, we prepared a non-responsive version to form supra-
molecular complexes as a contrast experiment and plotted the
catalytic activity versus temperature to obtain a contrastive
temperature curve under homogeneous conditions (Fig. 5A).
By replacing PNIPAM with PEG-5000, the contrastive supra-
molecular complexes comprising Ad-PEG, CD and CD-Se were
completely hydrophilic when the temperature increases from
25 1C to 43 1C. Thus the catalytic activity of complexes increased
slowly as temperature moved up and the maximum value only
reached 6.3 ꢀ 0.3 mM minꢁ1. Compared with our GPx mimic,
this result sufficiently demonstrated that the well-designed
artificial GPx mimic exhibited outstanding catalysis ability.
Furthermore, the catalytic rate (see Table 1) of our well-designed
GPx mimic showed two orders of magnitude bigger than PhSeSePh
under the identical conditions and exhibited considerable higher
catalytic activity. Meanwhile, refer to the catalytic activities that
were reported for the other systems.42–44 It was remarkable that our
GPx mimic proved at least two orders of magnitude larger than the
maximum catalytic rate of the selenium containing catalysts in
their systems (Table S1a and b, ESI†). The maximum catalytic rate
of our GPx mimic was determined to be similar to other artificial
enzymes (Table S1c, ESI† SGPx). However, SGPx was modified to
contain three catalytic elements (catalytic center), whereas our GPx
mimic has been modified to contain only one catalytic element. At
this point, our artificial selenium enzyme has been perceived as an
excellent GPx mimic with reasonably high catalytic activity.
Fig. 6 Plot of the initial rate at different concentrations of H2O2 in the
presence of the GPx mimic (2.0 mM catalytic center) at pH = 7.0 (50 mM
PBS). The initial concentration of GSH was fixed to 1 mM. The concentra-
tions of H2O2 were 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1.0 mM, 2.5 mM
and 5 mM, respectively.
Fig. 7 Reversible switch of the catalytic activity of the GPx mimic during
heating–cooling cycles between 25 1C and 37 1C. The GPx activities
of supramolecular nanoenzymes were calculated from the reduction of
H2O2 by GSH.
PNIPAM scaffold, the catalytic activity of the thermosensitive
GPx mimic exhibited excellent reversibility regulated by temperature.
After multiple heating–cooling cycles between 25 1C and 37 1C,
the catalytic rate of the artificial GPx was still maintained steady
as shown in Fig. 7. These results indicated that the formation
of the vesicle-like self-assembled structure played a vital role
in the regulatory functions of the catalytic behaviour of the
artificial GPx.
Furthermore, we studied the saturation kinetics of the GPx
mimic in peroxidase-like reaction at individual concentrations
of the substrate H2O2 to find out that the nanoenzyme fitted
the typical saturation kinetics and exhibited real catalytic
behaviour (Fig. 6). In the coupled reductase assay system, in
the presence of the GPx mimic (2.0 mM catalytic center) at 37 1C
and pH = 7.0, according to the Michaelis–Menten equation, the
apparent kinetic parameters of the GPx mimic were acquired
(app)
(app)
(app) (app)
as K
= 391.98 M, k
= 18.97 minꢁ1 and k
/KmGSH =
cat
mGSH
cat
Conclusions
4.84 ꢂ 104 mMꢁ1 minꢁ1 and the turnover number per catalytic
center of selenium was calculated to be 19 minꢁ1. This was taken In conclusion, through the amphipathic molecule multilevel
as proof that we have built a real enzyme mimic. Based on a self-assembly we have successfully constructed a temperature
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tive GPx mimic formed by the cyclodextrin-based host–guest
chemistry. The catalytic activity of the GPx mimic exhibited
reversibly regulated temperature-responsive characteristics.
Taken as a real enzyme catalyst, we also demonstrated its typical
saturation kinetics behaviour. At 25 1C, the catalytic activity of
the GPx mimic was found to be 1.3 ꢀ 0.08 (mM minꢁ1), in
contrast the value rapidly increased to 19.9 ꢀ 0.6 (mM minꢁ1
)
at 37 1C. The certain advantages for our research were that we
achieved facile preparation by noncovalent interactions and
controllable catalytic activity based on the assembly and dis-
assembly of the vesicle structure which provided a unique
microenvironment for enzymatic reaction near the body tem-
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Foundation of China (no. 21234004, 21420102007, 21574056,
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