A supramolecular microgel glutathione peroxidase mimic with temperature responsive activity

Article abstract of DOI:10.1039/c3sm53117a

Glutathione peroxidase (GPx) protects cells from oxidative damage by scavenging surplus reactive oxygen species (ROS). Commonly, an appropriate amount of ROS acts as a signal molecule in the metabolism. A smart artificial GPx exhibits adjustable catalytic activity, which can potentially reduce the amount of ROS to an appropriate degree and maintain its important physiological functions in metabolism. To construct an optimum and excellent smart artificial GPx, a novel supramolecular microgel artificial GPx (SM-Te) was prepared based on the supramolecular host-guest interaction employing the tellurium-containing guest molecule (ADA-Te-ADA) and the cyclodextrin-containing host block copolymer (poly(N-isopropylacrylamide)-b-[polyacrylamides-co-poly(6-o-(triethylene glycol monoacrylate ether)-β-cyclodextrin)], PPAM-CD) as building blocks. Subsequently, based on these building blocks, SM-Te was constructed and the formation of its self-assembled structure was confirmed by dynamic light scattering, NMR, SEM, TEM, etc. Typically, benefitting from the temperature responsive properties of the PNIPAM scaffold, SM-Te also exhibited similar temperature responsive behaviour. Importantly, the GPx catalytic rates of SM-Te displayed a noticeable temperature responsive characteristic. Moreover, SM-Te exhibited the typical saturation kinetics behaviour of a real enzyme catalyst. It was proved that the changes of the hydrophobic microenvironment and the pore size in the supramolecular microgel network of SM-Te played significant roles in altering the temperature responsive catalytic behaviour. The successful construction of SM-Te not only overcomes the insurmountable disadvantages existing in previous covalent bond crosslinked microgel artificial GPx but also bodes well for the development of novel intelligent antioxidant drugs. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3sm53117a

Volume 10 Number 19 21 May 2014 Pages 3349–3514
Soft Matter
www.softmatter.org
ISSN 1744-683X
PAPER
Yanzhen Yin, Shufei Jiao et al.
A supramolecular microgel glutathione peroxidase mimic with
temperature responsive activity

Soft Matter
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PAPER
A supramolecular microgel glutathione peroxidase
mimic with temperature responsive activity
Cite this: Soft Matter, 2014, 10, 3374
ab
a
Shufei Jiao, Chao Langc and Junqiu Liuc
*
*
Yanzhen Yin,
Glutathione peroxidase (GPx) protects cells from oxidative damage by scavenging surplus reactive oxygen
species (ROS). Commonly, an appropriate amount of ROS acts as a signal molecule in the metabolism. A
smart artificial GPx exhibits adjustable catalytic activity, which can potentially reduce the amount of ROS
to an appropriate degree and maintain its important physiological functions in metabolism. To construct
an optimum and excellent smart artificial GPx, a novel supramolecular microgel artificial GPx (SM-Te)
was prepared based on the supramolecular host–guest interaction employing the tellurium-containing
guest molecule (ADA-Te-ADA) and the cyclodextrin-containing host block copolymer (poly(N-
isopropylacrylamide)-b-[polyacrylamides-co-poly(6-o-(triethylene
glycol
monoacrylate
ether)-b-
cyclodextrin)], PPAM-CD) as building blocks. Subsequently, based on these building blocks, SM-Te was
constructed and the formation of its self-assembled structure was confirmed by dynamic light scattering,
NMR, SEM, TEM, etc. Typically, benefitting from the temperature responsive properties of the PNIPAM
scaffold, SM-Te also exhibited similar temperature responsive behaviour. Importantly, the GPx catalytic
rates of SM-Te displayed a noticeable temperature responsive characteristic. Moreover, SM-Te exhibited
the typical saturation kinetics behaviour of a real enzyme catalyst. It was proved that the changes of the
hydrophobic microenvironment and the pore size in the supramolecular microgel network of SM-Te
played significant roles in altering the temperature responsive catalytic behaviour. The successful
construction of SM-Te not only overcomes the insurmountable disadvantages existing in previous
covalent bond crosslinked microgel artificial GPx but also bodes well for the development of novel
intelligent antioxidant drugs.
Received 15th December 2013
Accepted 9th February 2014
DOI: 10.1039/c3sm53117a
www.rsc.org/softmatter
considerable efforts have been devoted to produce organo-
selenium/tellurium compounds that mimic the properties of
GPx in recent years.6–16 In our group, based on the under-
standing of the structure of a native GPx, some articial GPxs
have been designed employing the imprinting method,13
ATRP17,18 and blending process.19,20
Introduction
Reactive oxygen species (ROS) (hydrogen peroxides, organic
hydroperoxides and phospholipid hydroperoxides) are the
byproducts of cellular metabolism, which are normally harm-
less to humans and act as signal molecules in the metabolism.1
However, surplus ROS can lead to many human oxidative stress-
related diseases such as reperfusion injury, inammatory
process, neuronal apoptosis and cancer.2,3 Generally, such
oxidative stress-related diseases are controlled by the anti-
oxidative defense system, especially by the antioxidative enzyme
system. Especially, as a member of the family of antioxidative
enzymes, glutathione peroxidase (GPx, Ec.1.11.1.9) is an
important selenium-containing enzyme, which catalyzes the
reduction of hydroperoxides (ROOHs) using glutathione (GSH)
as the substrate.4,5 Owing to its biologically crucial role,
A microgel is a cross-linked polymer particle, which has been
rst described by Staudinger.21 Recently, polymer microgels
have been proven to be excellent scaffolds for biomaterials.22,23
Due to the unique property of combing the characteristics of a
linear macromolecule and a three-dimensional network, poly-
mer microgels have been widely used in the elds of articial
enzymes,22,24 regenerative medicines,25 sensors,26 drug delivery
systems,27 and so on. Especially, the microgel is one of the most
suitable scaffolds for an articial enzyme as its three-dimen-
sional space network is similar to the folding secondary struc-
ture of the native enzyme. Therefore, various articial enzymes
are constructed based on microgels.22,24,28,29 In our previous
report, we have successfully constructed a covalent bond
crosslinked microgel articial GPx based on a temperature-
responsive microgel using poly(N-isopropylacrylamide) (PNI-
PAM) as the scaffold (this smart articial GPx is denoted as
Microgel GPx in this report).24 Considering that Microgel GPx
aSchool of Chemistry and Chemical Engineering, Qinzhou University, No. 89, Xihuan
Nanlu, Qinzhou 535000, People's Republic of China. E-mail: yinyanzhen2013@163.
com; jiaoshufei2013@163.com; Fax: +86-0777-2860226
bGuangxi Experiment Centre of Science and Technology, Guangxi University, Nanning
530004, China
cState Key Laboratory of Supramolecular Structure and Materials, Jilin University,
Changchun 130012, People's Republic of China
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exhibited controllable catalytic activity, it could potentially be smart articial GPx, SM-Te displayed a noticeable temperature
applied to exploring intelligent antioxidant drugs for adjusting responsive catalytic activity and the typical saturation kinetics
the appropriate concentration of ROS as only surplus ROS could behaviour of a real enzyme catalyst. To the best of our knowl-
result in various illnesses. However, there were still two serious edge, SM-Te constructed in this work is the rst example of a
and insurmountable disadvantages in Microgel GPx. On the one smart articial GPx based on the scaffold of a supramolecular
hand, the synthetic process of the covalent bond crosslinked microgel. We anticipate that this articial GPx not only bodes
microgel was carried out employing cetyl trimethylammonium well for exploration of intelligent antioxidant drugs but also
bromide (CTAB) as the surfactant. The efficient separation of highlights the application of host–guest self-assembled supra-
CTAB from the microgel solution during the purication molecular materials.
process was not achievable. On the other hand, the synthetic
polymers with higher molecular weight can commonly elicit a
strong immune response and are difficult to be degraded or
excreted by the metabolic system when they were potentially
used as scaffolds for smart antioxidant drugs.30–32 Herein,
Experimental section
Materials
Tris(2-dimethylaminoethyl)amine (Me6TREN) was synthesized as
described previously.54 N-Isopropylacrylamide (NIPAM, Aldrich)
was recrystallized from hexane and toluene, and dried under
vacuum prior to use. Sodium borohydride and 3-bromo-1-prop-
anol were purchased from Fluka and were used without further
purication. Cumene hydroperoxide (CUOOH), H2O2, and NBT
were purchased from J&K Scientic Ltd and were used without
further purication. 3-Carboxyl-4-nitrobenzenethiol (TNB) was
synthesized from 5,50-dithiobis(2-nitrobenzoic acid) as described
previously.12 Acrylamide, b-cyclodextrin, triethylamine, tetrahy-
drofuran, phenyl methanol and 4-toluene sulfonyl chloride were
purchased from Shanghai Reagent Co. Acryloyl chloride and
2-bromopropionyl bromide were purchased from Anhui Wotu
Reagent Co. Triethylamine and tetrahydrofuran were rigorously
dried with sodium. 1-[p-(Phenyl-azo) phenoxyethyl]pyridinium
bromide (AZO) was endowed from Liu's group.
Microgel GPx exhibited the average diameter of 160 nm and its
molecular weight was far higher than that of the traditional
single chain polymer. Thus, this microgel was difficult to be
degraded or excreted by the metabolic system. These two
disadvantages have largely limited the further application of
Microgel GPx. Therefore, how to overcome these two insur-
mountable disadvantages, design a novel smart articial GPx,
and provide an optimum and excellent scaffold for the devel-
opment of antioxidant drugs are still great challenges.
Since supramolecular chemistry has emerged as a fascinating
eld and is expanded into many interdisciplinary elds, versatile
supramolecular materials have been designed.33–38 Among these
materials, supramolecular hydrogels are widely investigated. Due
to the unique structure formed through non-covalent interactions,
supramolecular hydrogels exhibit a reversible dynamic formation
process,39,40 and are extensively applied in the elds of sensors,41,42
controlled release systems,43,44 articial enzymes45,46 as well as self-
healing materials.47,48 These materials are also endowed with
potential degradability due to the unique property of non-covalent
interactions in supramolecular hydrogels. Amazingly, compared
with supramolecular hydrogels, supramolecular microgels exhibi-
ted the additional advantages of low solution viscosity and
nano-sized aggregation morphology, which provided an excellent
scaffold for nano-sized biomaterials.28,49,50 Commonly, the
construction of supramolecular microgels can be easily achieved,
which is like the ‘stacker game’ using functional building blocks.
Thus, desirable purity and productivity of the supramolecular
microgel are obtained. Moreover, considering that the structure of
the supramolecular microgel can be easily disassembled due to its
dynamic and reversible self-assembled properties, the biomate-
rials based on supramolecular microgels can be easily degraded
under appropriate conditions. Therefore, the supramolecular
microgel with these unique properties can be employed to over-
come the two insurmountable disadvantages existing in Microgel
Instrumentation
The characterization of the structures of the compounds was
performed with a Bruker 300 MHz spectrometer using a TMS
proton signal as the internal standard. UV-vis spectra were
obtained using a Pgeneral T6 UV-vis spectrophotometer. Scan-
ning electron microscopy (SEM) observations were carried out
using a JEOL JSM-6700F scanning electron microscope with a
primary electron energy of 3 kV. Transmission electron
microscopy (TEM) observations were carried out using a JEOL
JEM 3010 scanning electron microscope. The buffer pH values
were determined with a METTLER TOLEDO 320 pH meter.
Dynamic light scattering experiments were performed using a
Malvern ZETAS12-ERNANOSERIES instrument. Molecular
weights and molecular weight distributions were determined by
GPC using THF as the eluent at a ow rate of 1.0 mL minꢀ1
.
Characterization of ADA-Te-ADA, AZO and CD-monomer
GPx. To construct the excellent smart articial GPx with adjustable ADA-Te-ADA was synthesized according to the preparation
catalytic activity, effective preparation process and potential method of bis-(3-acryloyloxypropyl)-telluride in our previous
degradable ability, the combination of the articial GPx and the report except that acryloyl chloride was replaced by 1-ada-
supramolecular microgel is desirable.
mantanecarbonyl chloride.24 AZO was endowed from Liu's group.
Herein, the novel supramolecular microgel articial GPx The cyclodextrin containing a host monomer (6-o-(triethylene
(SM-Te) was crosslinked by the host–guest interaction between glycol monoacrylate ether)-b-cyclodextrin, CD-monomer) was
cyclodextrin and adamantine. Such a host–guest interaction has synthesized according to our previous report.19 The structures of
been proved to be the efficient non-covalent interaction for the them were determined using a Bruker 300 MHz spectrometer
construction of supramolecular materials.47,51–53 As a novel using a TMS proton signal as the internal standard.
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ADA-Te-ADA. 1H NMR (300 MHz, CDCl3) d (ppm) 4.09 (t, 2H),
Paper
phosphate buffer (pH ¼ 7.0. 50 mM) and 100 mL of the SM-Te
(10 mM) were added, and then 100 mL of the TNB solution
(1.5 mM) was added. The mixture in the quartz cuvette was pre-
incubated at appropriate temperature for 3 min. Finally, the
reaction was initiated by the addition of 100 mL of cumene
hydroperoxide (CUOOH) (2.5 mM), and the absorption decrease
of TNB at 410 nm (3410 ¼ 13 600 Mꢀ1 cmꢀ1, pH ¼ 7.0) was
monitored using a Pgeneral T6 UV-vis spectrophotometer.
Appropriate control of the non-enzymatic reaction was per-
formed and was subtracted from the catalyzed reaction.
2.66 (t, 2H), 2.07 (m, 2H), 2.01 (s, 3H), 1.88 (s, 6H), 1.71 (s, 6H).
AZO. 1H NMR (300 MHz), (CD3)2SO d (ppm) 9.17 (d, 2H), 8.66
(t, 1H), 8.21 (t, 2H), 7.90–7.81 (m, 4H), 7.60–7.52 (m, 3H), 7.13
(d, 2H), 5.10 (t, 2H), 4.65 (t, 2H).
CD-monomer. 1H NMR (300 MHz, D2O) d (ppm) 8.02 (s, 1H),
5.95–6.41 (m, 3H), 5.15 (s, 2), 5.00 (d, 7), 4.29 (t, 2H), 3.45–3.90
(m 56H (14H of glycol and 42H of cyclodextrin)).
Synthesis of PPAM-CD
The macroinitiator PNIPAM-Br and the cyclodextrin-containing
host block copolymer (poly(N-isopropylacrylamide)-b-[poly-
acrylamides-co-poly(6-o-(triethylene glycol monoacrylate ether)-
b-cyclodextrin)], PPAM-CD) were synthesized according to our
previous report.20 GPC analysis of PNIPAM-Br revealed a Mn of
8036 and a polydispersity, Mw/Mn, of 1.18. The Mn of PPAM-CD
was 14 840. The polydispersity of PPAM-CD was 1.33. The
concentration of cyclodextrin in PPAM-CD was estimated to be
3.2 ꢁ 10ꢀ4 mmol mgꢀ1 according to the NMR analysis.
Investigation of the stability of SM-Te
The stability of SM-Te was assayed based on the catalytic activity
of SM-Te and the recycled SM-Te. Their catalytic activity was
determined using the same method mentioned above (section
of Determination of GPx activity). Typically, the recycling
process of SM-Te and determination of catalytic activity were as
follows: rst, the catalytic reaction for the reduction of cumene
hydroperoxide (CUOOH) by 3-carboxyl-4-nitrobenzenethiol
(TNB) was carried out in a 1 mL quartz cuvette using the
traditional method (see Determination of GPx activity).
Secondly, the solution of SM-Te and other compounds of the
assay system in a 1 mL quartz cuvette was recycled. The solvent
of this solution was removed using a rotary evaporator and the
recycled compound (SM-Te and other compounds of the assay
LCST determination of PPAM-CD
The determination of LCST was carried out according to the
previously reported method.64 Typically, the optical trans-
mittance of the PPAM-CD solution (1 mg mLꢀ1) at different
temperatures was measured at 600 nm using a Pgeneral T6
UV-vis spectrophotometer. Sample cells were thermostatted in a
circulator bath at different temperatures from 25 to 45 ꢂC prior
to the measurements. The LCST was dened as the temperature
of the 50% transmittance point during the rst heating ramp.
ꢂ
system) was dried under vacuum for 24 h at 45 C. Then, the
catalytic activity of such recycled SM-Te was determined except
that SM-Te was replaced by the recycled SM-Te. Subsequently,
the catalytic composition in the assay system was recycled once
again using a similar method mentioned above, and the cor-
responding catalytic activity was determined once again. A
similar recycling process and determination of catalytic activity
were repeated for a total of three times. The catalytic rates are
illustrated in Fig. 9.
ꢂ
The LCST of PPAM-CD was 34.4 C.
Preparation of supramolecular microgel SM-Te
Deionized water (9.5 mL) was introduced into a 25 mL ask.
PPAM-CD (31.3 mg, 0.01 mmol) was added and dissolved in this
solution. ADA-Te-ADA (2.85 mg, 0.005 mmol) was dissolved in
methanol (0.5 mL). The solution of PPAM-CD was thermo-
Results and discussion
Construction of supramolecular microgel SM-Te
ꢂ
statted in a circulator bath at 35 C (above the LCST of PPAM-
CD) for 10 min. Then, the methanol solution of ADA-Te-ADA
was slowly added into the solution of PPAM-CD under sonica-
tion at 35 ꢂC. Aer the dropwise process was nished, the
mixture solution was treated under continual sonication at
35 ꢂC for 20 min. Then, the supramolecular microgel SM-Te was
obtained with a concentration of 3.42 mg mLꢀ1. The concen-
tration of tellurium (catalytic center) was 0.5 mM.
Commonly, ROS are harmless to humans and act as signal
molecules in the metabolism. However, the overproduction of
ROS can cause many human oxidative stress-related diseases.
Therefore, in our previous report, a smart articial GPx
(Microgel GPx) with controllable catalytic activity was con-
structed based on the covalent bond crosslinked microgel
(PNIPAM) scaffold, which was designed to endow Microgel GPx
with the temperature responsive catalytic activity. It can
potentially be used in the development of intelligent antioxi-
dant drugs to adjust the appropriate concentration of ROS.24
Although the successful construction of Microgel GPx high-
lights the development of intelligent antioxidant drugs, there
are serious and insurmountable disadvantages in Microgel GPx
related to its purication process and covalently crosslinked
scaffold. Firstly, the efficient separation of CTAB from the
LCST determination of SM-Te
The determination of optical transmittance of the SM-Te (1 mg
mLꢀ1) solution at different temperatures was performed using a
similar method to that of PPAM-CD. The LCST of SM-Te was
ꢂ
32.8 C.
Determination of GPx activity
The catalytic activity was assayed according to a modied microgel solution during the purication process was not
method reported by Hilvert et al.6 Typically, the reaction was achievable. Secondly, the covalently crosslinked microgel scaf-
carried out at 25 ꢂC in a 1 mL quartz cuvette, 700 mL of fold of Microgel GPx exhibited an average diameter of 160 nm.
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And its molecular weight was far higher than that of the tradi-
tional single chain polymer. Therefore, Microgel GPx was diffi-
cult to be degraded or excreted by the metabolic system. These
two disadvantages have largely limited the further application
of Microgel GPx. Usually, the purity and productivity of the
supramolecular microgel is desirable. Importantly, as the
supramolecular microgel has the unique property of dynamic
and reversible self-assembled behaviour, the articial GPx
based on the supramolecular microgel can be easily degraded
under appropriate conditions. Therefore, SM-Te can be
endowed with the advantages of the effective preparation
process and potential degradable ability if SM-Te was con-
structed based on the supramolecular microgel scaffold. In
other words, compared with previous Microgel GPx, SM-Te was
more advantageous, the successful construction of which will
overcome the two insurmountable disadvantages mentioned
above. Herein, to construct the supramolecular scaffold of SM-
Te, the corresponding supramolecular building block was
designed. As shown in Fig. 1, the monomer NIPAM, acrylamide
(AM), and CD-monomer were selected as functional monomers.
Herein, NIPAM endowed the scaffold of SM-Te with
temperature responsive behaviour. AM functions as the hydro-
philic block in the scaffold of PPAM-CD. It is known that the
soluble PNIPAM can transform into an insoluble polymer when
the temperature becomes higher than LCST. The soluble block
copolymer bearing the PNIPAM block can transform into an
amphiphilic polymer when the temperature becomes higher
than LCST. Under these conditions, the controllable aggregated
structures (micelles, vesicles, nanorods and so on) can be
formed. Therefore, AM acted as the hydrophilic block in the
amphiphilic PPAM-CD when the temperature was above the
LCST, which provides the platform for the construction of SM-
Te. The graphical representation of the self-assembly process
(A–C) is shown in Scheme 1. The CD-monomer functions as the
host molecule in PPAM-CD, which can be further employed to
complex the guest molecule in the construction process of SM-
Te. Considering that ATRP was an efficient technique for the
Fig. 1 The structures of the functional monomers NIPAM, AM, CD-
comonomer, cyclodextrin-containing host block copolymer (PPAM-
CD), tellurium-containing guest molecule (ADA-Te-ADA) and
substrates (NBT, TNB).
synthesis of block copolymers with a controlled structure,55,56 to hydrophobic when the temperature was above the LCST.
PPAM-CD was synthesized by ATRP and used as the building Therefore, the soluble block copolymer bearing the PNIPAM
block for SM-Te. Additionally, the crystal structure of bovine block can transform into an amphiphilic polymer when the
erythrocyte GPx was reported by Epp et al. in 1983.5 The catalytic temperature was above the LCST, which provides the platform
active site of GPx has been well studied by mimicking the for the construction of versatile self-assembled materials.24,59,60
catalytic center of selenocysteine in GPx using tellurium-con- Benetting from the temperature responsive properties of the
taining molecules.17,32 It has been proved that tellurium-con- PNIPAM scaffold, Microgel GPx exhibited the temperature
taining molecules act as more efficient scaffolds for articial responsive catalytic behaviour during the temperature respon-
GPx compared with selenium-containing molecules. Therefore, sive process of the PNIPAM scaffold. Herein, the temperature
the tellurium-containing guest molecule (ADA-Te-ADA) was responsive properties of the PNIPAM scaffold in PPAM-CD were
synthesized, which served two purposes in SM-Te: one as an also employed in the construction of SM-Te, which might
excellent alternative for selenocysteine in native GPx and the provide the basement for adjusting the catalytic activity of SM-
other one as a cross-linker for the construction of SM-Te based Te. To provide the basic information for the construction of SM-
on the host–guest interaction. Additionally, AZO was employed Te, the temperature responsive properties of PPAM-CD were rst
as a competitive guest for the conrmation of the formation of invesꢂtigated. The LCST of PPAM-CD was determined to be
the supramolecular microgel in the NMR assay.
34.4 C (shown in Fig. 2a). The stable optical transmittance of
As PNIPAM is a thermally sensitive polymer with a lower PPAM-CD was observed when the temperature was higher than
critical solution temperature (LCST) of 32 ꢂC, it undergoes a 35 ꢂC.20 Additionally, from Fig. 3a, the hydrodynamic diameter of
reversible volume phase transition at near-physiological PPAM-CD at 25 ꢂC was less than 10 nm, which proved that
temperature.57,58 Its polymer block shis from being hydrophilic PPAM-CD was soluble in water. However, the self-assembled
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Paper
Scheme 1 A graphical representation of the self-assembled process (A–C) and the reversible responsive behaviour (C and D) of SM-Te.
aggregation with 230 nm (Fig. 3d) in hydrodynamic diameter conrmation of the formation of SM-Te is given in the following
was formed at 35 ꢂC as PPAM-CD was transformed into an section.
amphiphilic polymer. These observations indicated that the
ꢂ
efficient self-assembled aggregation could be formed at 35 C.
Therefore, SM-Te was prepared at 35 ꢂC and the graphical
Characterization of supramolecular microgel SM-Te
The formation of the host–guest supramolecular complex in the
network of SM-Te was conrmed by the change of optical
transmittance, dynamic light scattering, NMR, SEM, and TEM.
From Fig. 2, it can be found that the temperature dependence of
optical transmittance of SM-Te (curve b) was different from that
representation of the self-assembly process is given in Scheme 1.
As illustrated in Scheme 1A, PPAM-CD was soluble at 25 ꢂC.
Then, as shown in Scheme 1B, the self-assembled aggregation
was formed and the distance of cyclodextrins in PPAM-CD was
decreased when the temperature was 35 ꢂC which was higher
than the LCST of PPAM-CD. Subsequently, the guest molecule
ADA-Te-ADA was added into the above solution under sonication
at 35 ꢂC. Accordingly, the host–guest supramolecular complex in
the network of SM-Te was formed between cyclodextrin in PPAM-
CD and adamantane in ADA-Te-ADA (Scheme 1C). The
ꢂ
of PPAM-CD (curve a). The LCST of SM-Te was 32.8 C, which
ꢂ
was lower than that of PPAM-CD (34.4 C). We supposed that
two factors played roles in the decrease of LCST. On the one
hand, the network of SM-Te was crosslinked by ADA-Te-ADA.
The molecular morphology of SM-Te with a network structure
was larger than that of PPAM-CD (the normal single chain
polymer). It has been proved that the aqueous solution of
PNIPAM showed a strong decrease of LCST with increasing
molecular weight.57,58 Therefore, the decreased LCST of SM-Te
possibly resulted from that the molecular weight of SM-Te was
higher than that of PPAM-CD. One the other hand, considering
Fig. 3 Hydrodynamic diameters of PPAM-CD at varying temperatures
Fig. 2 Temperature dependence of optical transmittance at 600 nm (a, 25 ꢂC; d, 35 ꢂC) and hydrodynamic diameters of SM-Te at varying
obtained for solutions (pH ¼ 7.0, 50 mM PBS) of (a) PPAM-CD and (b) temperatures (b, 45 ꢂC; c, 35 ꢂC; e, 25 ꢂC) determined using a Malvern
SM-Te at concentrations of 1 mg mLꢀ1
.
ZETAS12-ERNANOSERIES instrument.
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that ADA-Te-ADA was a hydrophobic molecule and acted as the
crosslinker in SM-Te, SM-Te was anchored with more hydro-
phobic factors and might exhibit lower LCST. This phenom-
enon was in good agreement with
a
previous report.61
Consequently, the difference of the LCSTs between SM-Te and
PPAM-CD preliminarily proved that the self-assembled struc-
ture of SM-Te was formed. Additionally, comparing the
temperature dependence of optical transmittance of SM-Te and
Microgel GPx,24 we found that they exhibited similar tempera-
ture responsive behaviour. Considering that the change of the
structure can result in the change of catalytic activity of an
articial enzyme, similar temperature responsive behaviour of
SM-Te and Microgel GPx might enable their catalytic activities
exhibiting similar temperature responsive behaviour. The
conrmation of this suspect is presented in the following
section (Catalytic activity modulated by temperature).
To further prove the formation of the host–guest complex in
SM-Te, NMR assay was employed using AZO as the competitive
guest. Compared with the host–guest interaction between AZO
and cyclodextrin, the host–guest interaction between ADA-Te-
ADA and cyclodextrin was much stronger. The host–guest
complex between ADA-Te-ADA and cyclodextrin was more
stable. Therefore, we assumed that ADA-Te-ADA could supplant
AZO from the cave of cyclodextrin even if the inclusion
complexation had primarily formed between AZO and cyclo-
dextrin. In other words, AZO could act as the indicator to
Fig. 4 1H NMR spectra of (A) the proton signals of aromatic rings in
pure AZO, (B) the proton signals of aromatic rings in the binary system
of AZO/cyclodextrin, and (C) the proton signals of aromatic rings in the
ternary system of AZO/cyclodextrin/ADA-Te-ADA in D2O.
conrm the host–guest complex between ADA-Te-ADA and of d2 and g2 shied to a high eld (proton signals of e2 and f2
cyclodextrin. The graphical representation of the competitive were buried by the protons of the polymer), which suggested
complex mechanism using AZO as an indicator is shown in that these protons were included in the hydrophobic cavity of
Scheme 2.
cyclodextrin and the proton signals were shielded. These results
To conrm this hypothesis, three groups of 1H NMR spectra were in line with the previous report that investigated the
are characterized and illustrated in Fig. 4. Among three groups formation mechanism of the host–guest complex between AZO
of 1H NMR spectra, Fig. 4A provides the proton signals of and functional cyclodextrin.62 Moreover, proton signals of d2
aromatic rings in pure AZO, Fig. 4B provides the proton signals and g2 were broad peaks compared with d1 and g1, which also
of aromatic rings in the binary system of AZO/cyclodextrin, and proved that these protons were included in the hydrophobic
Fig. 4C provides the proton signals of aromatic rings in the cavity of cyclodextrin. A similar result was also reported previ-
ternary system of AZO/cyclodextrin/ADA-Te-ADA. Comparing ously.63 Subsequently, a ternary system of AZO/cyclodextrin/
Fig. 4B with Fig. 4A, we noticed that proton signals of c2, a2, b2, ADA-Te-ADA was obtained by adding ADA-Te-ADA into the
h2 shied to a low eld, which suggested that these protons in above binary system of AZO/cyclodextrin (as shown in Fig. 4C).
the binary system were exposed to water moderately and were It was noticeable that the proton signals of aromatic rings in the
not included by cyclodextrin. We also found that proton signals ternary system were the same as that of pure AZO, which sug-
gested that the AZO was not included in the cavity of cyclo-
dextrin and the complex of AZO/cyclodextrin was not formed.
Equally, it conrmed the hypothesis that ADA-Te-ADA could
supplant AZO from the cave of cyclodextrin and the complex of
ADA-Te-ADA/cyclodextrin was indeed formed.
As the supramolecular microgel was used as the scaffold for
SM-Te, it was necessary to reveal the detailed information of the
aggregation morphology that was essential for the investigation
of the catalytic mechanism of articial GPx. Firstly, dynamic
light scattering was employed to explore the temperature
dependence of the hydrodynamic diameter of SM-Te. As shown
in Fig. 3, the hydrodynamic diameters of SM-Te at 25 ꢂC (curve
e), 35 ꢂC (curve c), and 45 ꢂC (curve b) were 360 nm, 190 nm, and
98 nm, respectively. Especially, it was noticeable that the
ꢂ
hydrodynamic diameter of SM-Te (Fig. 3e) at 25 C (below the
LCST of 32.8 ꢂC) was strikingly different from that of PPAM-CD
Scheme 2 A graphical representation of the competitive complex
mechanism using AZO as an indicator.
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(Fig. 3a) under the same conditions. The difference in the
diameters was possibly derived from the different molecular
morphologies of SM-Te and PPAM-CD. It further proved that the
crosslinked supramolecular microgel with a larger hydrody-
namic diameter was formed. Additionally, the hydrodynamic
diaꢂmeter was decreased with the increase of temperature from
ꢂ
35 C to 45 C, which might be caused by the reason that PNI-
PAM in SM-Te became more hydrophobic and the network
scaffold of SM-Te was contracted at higher temperatures.
Commonly, the change of the hydrophobicity of PNIPAM was
caused by the break of hydrogen bonds between the amide
group of PNIPAM and the surrounding water. For naturally
occurring enzymes, abundant studies indicated that a minor
change of the structure of the enzyme would result in a
dramatic change in catalytic activity. Herein, the temperature
responsive changes of the hydrophobic diameter of SM-Te
provided important insight into the regulation of the catalytic
activity of smart articial GPx.
Furthermore, the actual morphology of SM-Te when the
temperature was higher than its LCST was observed by SEM
(as shown in Fig. 5). From the SEM image, we clearly found that
the presence of spherical nanoparticles of about 80 nm in
average diameter. The dimensions of the nanoparticles
observed from SEM were smaller than those obtained from
dynamic light scattering. This phenomenon might be derived
from the reason that the zetasizer nano-instrument reported the
average hydrophobic diameter with the contribution of a
swollen corona of nanoparticles. To further reveal the detailed
morphologies of the spherical nanoparticles, their structures
were observed by TEM (as shown in Fig. 6). It was apparent that
the diameters of spherical nanoparticles were 70–80 nm, which
was in close agreement with the SEM assay. Particularly, many
tiny holes were observed in the spherical nanoparticles, which
was the typical character of a microgel. In our previous report,
the nanoparticles assembled by PPAM-CD were hollow vesicle-
like nanoparticles with a thinner region surrounded by a thicker
region.20 Herein, the unique structure of SM-Te observed by
TEM further proved that the supramolecular microgel was
indeed prepared.
Fig. 6 TEM image of SM-Te.
Consequently, all the results based on optical transmittance,
dynamic light scattering, NMR, SEM, and TEM indicated that
the supramolecular microgel SM-Te was successfully con-
structed. Additionally, comparing the detailed information of
the structure of SM-Te mentioned above and that of Microgel
GPx,24 we concluded that they exhibited similar nano-scaled
aggregated structures. It is known that a slight change of the
structure will result in a dramatic change in catalytic activity of a
successful articial enzyme. Considering that SM-Te and
Microgel GPx exhibited the structures with a similar aggregated
scale, we anticipated that they might be endowed with similar
catalytic behaviour. The conrmation of this suspect is pre-
sented in the following section (Catalytic behaviour of supra-
molecular microgel SM-Te).
Finally, the graphical representations of the self-assembled
process of SM-Te (Scheme 1A–C) and the reversible responsive
behaviour of SM-Te (Scheme 1C and D) are illustrated.
Catalytic behaviour of supramolecular microgel SM-Te
Herein, for evaluating the catalytic behaviour of SM-Te, the
catalytic activity of SM-Te for the reduction of cumene hydro-
peroxide (CUOOH) by TNB (3-carboxyl-4-nitrobenzenethiol) was
determined according to a modied method reported by Hilvert
et al.6 using TNB as a GSH alternative (see Fig. 7). The activity
was given assuming one molecule catalytic center (Te-mono-
mer) in SM-Te as one active site of the enzyme. The reaction was
initiated by the subsequent addition of hydroperoxide, and the
absorbance at 410 nm (3410 ¼ 13 600 Mꢀ1 cmꢀ1, pH ¼ 7.0) was
recorded for a few minutes to calculate the reaction rate. The
relative activities are summarized in Table 1.
To investigate the temperature responsive properties of SM-
Te, the catalytic activity was determined in the TNB assay
system using CUOOH as the substrate at various temperatures
Fig. 7 Determination of the GPx catalytic rate for the reduction of
cumene hydroperoxide (CUOOH) using TNB (3-carboxyl-4-nitro-
benzenethiol) as the substrate.
Fig. 5 SEM image of SM-Te.
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ꢂ
ꢂ
from 25 C to 45 C (as shown in Table 1). To the best of our
knowledge, for the majority of temperature-activated reactions,
the reaction rates will enhance with the rising temperature
according to the Arrhenius equation. However, it was different
for SM-Te. To vividly present the temperature responsive
behaviour of SM-Te, a bell shaped thermally responsive catalytic
activity curve was obtained by plotting the catalytic reaction
rates against temperatures (Fig. 8). It was observed that the
catalytic rate increased slowly with incrꢂeasing temperature
when the temperature was lower than 32 C, and it increased
largely with the increase of the temperature from 32 ꢂC to 38 ꢂC.
Nevertheless, it was noticeable that the catalytic activity
decreased sharply when the temperature was further increased
above 38 ꢂC. Similarly, for the temperature responsive behav-
iour of Microgel GPx,24 a bell shaped curve was also obtained by
plotting the catalytic rates against temperature. Contrastively,
the highest catalytic rate was obtained at 32 ꢂC for Microgel GPx.
However, the highest catalytic rate was obtained at 38 ꢂC for SM-
Fig. 8 Plots of the catalytic rates of SM-Te versus temperatures during
the catalytic reduction of CUOOH (0.25 mM) by TNB (0.15 mM) and a
catalytic center (1.00 mM).
Te. Signicantly, the temperature of 38 ꢂC (related to the highest presented in the following section (Catalytic activity modulated
catalytic rate of SM-Te) was close to the physiological tempera- by temperature).
ture compared with 32 ꢂC (related to the highest catalytic rate of
Moreover, as a catalyst, the investigation of the stability of
Microgel GPx). Therefore, considering that the temperature of the composition of SM-Te prior to reaction and aer reaction is
the highest catalytic rate of SM-Te was close to the physiological really important. It is essential to the further investigation and
temperature, SM-Te was superior to Microgel GPx when it was application of SM-Te. It is known that a slight change of the
used to develop intelligent antioxidant drugs. Especially, for structure will result in a dramatic change in the catalytic activity
SM-Te, the highest catalytic rate was obtained at higher of an articial enzyme and a native enzyme. In other words, SM-
temperature, which might be derived from the stronger hydro- Te will exhibit a large change in catalytic activity if its compo-
philicity of the SM-Te scaffold as the hydrophilic cyclodextrin sition has changed. Therefore, considering that the catalytic
was anchored. Herein, for the temperature responsive catalytic activity was crucially important for a successful articial GPx,
behaviour of SM-Te, we speculated that such a phenomenon we chose the data of the catalytic activity as the effective values
was derived from the changes of the hydrophobic microenvi- to evaluate the stability of the composition of SM-Te. Herein, to
ronment and pore size of the supramolecular microgel network achieve this aim, we designed an experiment (see Investigation
by altering the temperature. The conrmation of this suspect is of the stability of SM-Te in the Experimental section). And the
catalytic activity of SM-Te, recycled SM-Te for one time, recycled
SM-Te for two times, and recycled SM-Te for three times was
5.56 mM minꢀ1, 5.49 mM minꢀ1, 5.44 mM minꢀ1, and 5.32 mM
minꢀ1, respectively. And the vivid histogram of catalytic rates is
illustrated in Fig. 9. By analysis of these catalytic rates, we found
that the catalytic activities were slightly changed during the
recycled process. Considering that a slight change of the
structure would result in a dramatic change in the catalytic
activity of an articial enzyme, the slight change of catalytic
activity indirectly proved that the composition of SM-Te was
stable under the reaction conditions and the recycled process of
SM-Te. Equally, it has been proved that the composition of
SM-Te was stable and remained the same prior to reaction and
aer reaction.
Commonly, the catalytic rate of the background (nonenzy-
matic) reaction between CUOOH and TNB at 38 ꢂC was very slow.
As shown in Table 1, a slight enhancement in the catalytic rate
was observed (v0 ¼ 0.011 mM minꢀ1) when the traditional small
molecule articial GPx (PhSeSePh) was used under the identical
conditions. Amazingly, SM-Te constructed in this work exhibited
a remarkable rate enhancement (v0 ¼ 5.60 mM minꢀ1). Compared
with the catalytic rate of previous Microgel GPx (see Table 1, v0 ¼
3.94 mM minꢀ1) determined under the same conditions, SM-Te
exhibited higher catalytic activity. Additionally, considering that
Table
1 The initial rates (v0) and activities for the reduction of
hydroperoxides (250 mM) by TNB (150 mM) in the presence of SM-Te at
pH 7.0 (50 mM PBS) and 36 ꢂC
a
Temperature
(ꢂC)
y0
Number Catalyst
ArSH ROOH
(mM minꢀ1
)
1
2
3
4
5
6
7
8
SM-Te
SM-Te
SM-Te
SM-Te
SM-Te
SM-Te
SM-Te
SM-Te
SM-Te
25
28
32
34
36
38
40
43
45
TNB CUOOH 2.31
TNB CUOOH 2.60
TNB CUOOH 3.12
TNB CUOOH 4.32
TNB CUOOH 5.08
TNB CUOOH 5.60
TNB CUOOH 5.26
TNB CUOOH 4.63
TNB CUOOH 3.82
TNB CUOOH 3.94
TNB CUOOH 0.011
9
10
11
12
13
14
Microgel GPxb 38
PhSeSePh
SM-Te
SM-Te
38
38
38
38
TNB H2O2
NBT CUOOH 6.25
NBT H2O2 1.28
0.86
SM-Te
a
The initial rate of reaction was corrected for the spontaneous
oxidation. And the concentration of the catalyst is 1 mM and assuming
one molecule catalytic center (tellurium moiety) as one active site of
b
the enzyme. The covalent bond crosslinked microgel articial GPx
constructed in our previous report.24
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a supramolecular microgel becoming smaller. It might further
result in the changes of the substrate binding ability and cata-
lytic activity. Thus, we speculated that the temperature
responsive catalytic behaviour of SM-Te was endowed from the
changes of the hydrophobic microenvironment and pore size of
the supramolecular microgel network. The graphical represen-
tation of responsive behaviour of the supramolecular microgel
network in SM-Te is illustrated in Scheme 1C and D. To expli-
cate the temperature responsive catalytic mechanism, optical
transmittance, dynamic light scattering and catalytic rates
measured in different assay systems were employed.
Based on the change of optical transmittance with increasing
temperature mentioned in Fig. 2b, we noticed that the optical
transmittance of SM-Te was stable (below 2%) when the
temperature was above 35 ꢂC. It meant that the efficient self-
assembled aggregation was formed above 35 ꢂC, which was
resulted from the thermal sensitive properties of the PNIPAN
scaffold in SM-Te. Commonly, the hydrophobic microenviron-
ment could be formed in the self-assembled aggregation when
the temperature was above the LCST of PNIPAN. Signicantly,
the hydrophobic microenvironment is one of the pivotal cata-
lytic factors that contribute to maintain efficient GPx catalytic
activity.19,24 Herein, this crucial role of the hydrophobic micro-
environment was reected by the remarkably enhanced catalytic
rates when the temperature increased from 32 ꢂC to 38 ꢂC.
Moreover, we proved that the pore size existed in a covalent
bond network of Microgel GPx played an important role in
altering the catalytic rates.24 Similarly, this phenomenon was
also observed in SM-Te. Combining the change of hydrody-
namic diameter with increasing temperature as shown in Fig. 3
and the change of catalytic rate with increasing temperature as
shown in Fig. 8, we found that the catalytic rates could be
adjusted according to the change of hydrodynamic diameter.
Typically, the hydrodynamic diameter was about 360 nm when
Fig. 9 The initial rates (v0) for the reduction of CUOOH (250 mM) by
TNB (150 mM) in the presence of SM-Te (a) and recycled SM-Te (b:
recycled for one time; c: recycled for two times; d: recycled for three
times).
SM-Te can overcome the insurmountable disadvantages existed
in previous microgel articial GPx, SM-Te was satisfactory as a
novel and excellent smart articial GPx. Moreover, as shown in
Fig. 10, the saturation kinetics behaviour of SM-Te for the
peroxidase reaction were studied at the individual concentrations
of CUOOH, which indicated that SM-Te exhibited typical satu-
ration kinetics behaviour and acted as a real catalyst for the
peroxidase reaction. In the TNB assay system, the apparent
kinetic parameters were obtained as follows: Vmax ¼ 16.32 mM
app
minꢀ1, k ¼ 16.32 minꢀ1, Km CUOOH ¼ 418.01 mM and kcaaptp/Km
cat
¼ 3.90 ꢁ 104 Mꢀ1 minꢀ1, and the turnover number per
CUOOH
catalytic center tellurium was calculated to be 16 minꢀ1
.
Comparing the apparent kinetic parameters of SM-Te mentioned
above and that of Microgel GPx,24 similar saturation kinetics
behaviour was observed. The similarity of saturation kinetics
behaviour might be derived from similar aggregated structures
and similar temperature responsive behaviour of their scaffolds.
Although SM-Te and Microgel GPx exhibited similar adjustable
catalytic activity and saturation kinetics behaviour, SM-Te was
endowed with additional advantages of effective preparation
process and potential degradable ability. Therefore, SM-Te was a
more excellent scaffold for the development of intelligent anti-
oxidant drugs.
ꢂ
ꢂ
the temperature was 25 C (below the LCST of 32.8 C). Under
these conditions, the microgel scaffold of SM-Te was swelling,
the optical transmittance was near 100%, the pore size of SM-Te
was larger, and the hydrophobic microenvironment was not
formed. Therefore, the pivotal catalytic factor was absent, which
Catalytic activity modulated by temperature
As shown in Fig. 8, SM-Te exhibited typical temperature
responsive catalytic behaviour. Herein, ADA-Te-ADA was
anchored with the GPx catalytic center (tellurium) and acted as
the crosslinker for the supramolecular microgel network. In our
previous work, the covalent bond crosslinker with the GPx
catalytic center was employed, which was anchored into the
microgel scaffold of Microgel GPx.24 It was proved that the
change of the pore size of microgel networks of Microgel GPx,
the change of the hydrophobic microenvironment, and the
shrinkage of Microgel GPx play important roles in adjusting the
catalytic activity. Considering that SM-Te and Microgel GPx
were constructed based on a similar PNIPAM scaffold, the
Fig. 10 Plots of initial rates at different concentrations of CUOOH in
the presence of SM-Te. The initial concentration of TNB was fixed to
0.15 mM. The concentrations of CUOOH were 0.05, 0.10, 0.25, 0.5, 1,
shrinkage of a PNIPAM scaffold would result in the pore size of 2.5 and 5 mM, respectively.
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further resulted in the weak substrate binding ability and low
catalytic rate. Subsequently, the microgel scaffold of SM-Te was
deswelling when the temperature was 35 ꢂC (above the LCST of
32.8 ꢂC). The hydrodynamic diameter was about 190 nm, the
optical transmittance was below 2%, the pore size of SM-Te was
contracted, and the hydrophobic microenvironment was
preliminarily formed. As the pivotal catalytic factor that
contributes to maintain efficient GPx catalytic activity was pre-
sented, the substrate binding ability and catalytic rate were
remarkably enhanced under these conditions. However, as
shown in Fig. 8, it was noticeable that the catalytic activity
decreased sharply when the temperature continuously
increased above 38 ꢂC. Typically, under these conditions at
45 ꢂC, the hydrodynamic diameter was about 98 nm, the
hydrophobicity of the PNIPAM scaffold was further increased,
and the pore sizes in SM-Te were further contracted, which were
not suitable for allowing substrates to permeate into the cores
of SM-Te and nish the GPx catalytic reaction. Therefore, the
Fig. 11 The initial rates (v0) for the reduction of hydroperoxide (250
mM) by thiol TNB or NBT (150 mM) in the presence of SM-Te at pH 7.0
(50 mM PBS) and 38 ꢂC: (A) CUOOH, TNB; (B) CUOOH, NBT; (C) H2O2,
TNB; (D) H2O2, NBT.
catalytic rate was sharply decreased under these conditions as catalytic reaction. Whereas the NBT with a smaller size could
the efficient binding ability for substrates could not be ach- still permeate into the core to nish the GPx catalytic reaction,
ieved. These observations proved that the changes of the and the higher catalytic activity was obtained. This phenom-
hydrophobic microenvironment and pore size indeed played enon suggested that the effect of pore size was vital for adjusting
important roles in regulating the GPx catalytic rates. A similar the catalytic behaviour. Secondly, we also found that the higher
temperature responsive catalytic mechanism was also investi- catalytic rate was observed when CUOOH was used as the
gated for Microgel GPx.24
substrate (as shown in Fig. 11, A > C, or B > D). Commonly, the
Additionally, the maximum catalytic rate was obtained at 38 rate constants of the spontaneous reaction between hydroper-
ꢂC. The catalytic rates obtained at 35 ꢂC (the optical trans- oxide and thiol vary in magnitude in the order k(H2O2) >
mittance was just stable) and at LCST of SM-Te were all lower k(CUOOH).12 Nevertheless, it was noticeable that the higher
than the maximum catalytic rate. It was because that the catalytic rate was observed when CUOOH was used as the
ꢂ
optimum substrate binding ability was not achieved below 38 substrate for SM-Te at 38 C. Typically, the difference between
ꢂC even if the hydrophobic microenvironment and pore size are CUOOH and H2O2 was that CUOOH was a more hydrophobic
maintained to some extent under these conditions.
substrate than H2O2 owing to the presence of the hydrophobic
To further conrm that changes of the hydrophobic micro- cumenyl group in CUOOH. The difference in the catalytic rate in
environment and pore size of the supramolecular microgel different assay systems (the different activity for CUOOH and
network played important roles in adjusting the temperature H2O2) was mainly because that the hydrophobic microenvi-
responsive catalytic rates. The catalytic reactions were ronment enabled the hydrophobic substrate CUOOH to
measured in different assay systems using various substrates approach the catalytic center (tellurium in ADA-Te-ADA) effi-
with different hydrophobic abilities and molecule sizes ciently and complete the catalytic cycle more preferentially. It
(CUOOH, TNB assay system (Fig. 11A), v0 ¼ 5.60 mM minꢀ1
;
;
was proved that the different structure of CUOOH and H2O2
played an important role in different activities. Additionally,
CUOOH, NBT assay system (Fig. 11B), v0 ¼ 6.25 mM minꢀ1
H2O2, TNB assay system (Fig. 11C), v0 ¼ 1.28 mM minꢀ1; H2O2, this observation also proved that hydrophobic microenviron-
NBT assay system (Fig. 11A), v0 ¼ 0.86 mM minꢀ1). The differ- ment in SM-Te also played an important role in adjusting the
ence between NBT and TNB was that NBT was a smaller mole- temperature responsive catalytic behaviour.
cule in size than TNB owing to the lack of a carboxyl functional
Therefore, based on the discussions mentioned above, we
group in NBT. The difference between CUOOH and H2O2 was could draw a conclusion that both the changes of the hydrophobic
that CUOOH was a more hydrophobic substrate than H2O2 microenvironment and pore size in the supramolecular microgel
owing to the presence of the hydrophobic cumenyl group in network of SM-Te played signicant roles in adjusting the
CUOOH.
temperature responsive catalytic behaviour. SM-Te and Microgel
Firstly, under the identical conditions at 38 ꢂC, we found that GPx exhibited similar temperature responsive catalytic activity
the higher catalytic rate was observed when NBT was used as the and saturation kinetics behaviour. Especially, SM-Te exhibited a
substrate (as shown in Fig. 11, A < B, or C < D). Considering that higher catalytic rate than Microgel GPx under the same conditions
the hydrophobic microenvironment has formed and the pore at 38 ꢂC (nearing the physiological temperature). Signicantly,
size has contracted at 38 ꢂC, a dramatic change in the catalytic SM-Te was endowed with the advantages of effective preparation
rate in different assay systems using TNB or NBT as the process and potential degradable ability, which could overcome
substrate was anticipated. It should be caused by the shrinkage the two insurmountable disadvantages of Microgel GPx. Consid-
of the pore size in the network of SM-Te that made TNB not ering that SM-Te exhibited such unique advantages compared
easily permeate into the cores of SM-Te and nish the GPx with Microgel GPx, SM-Te was a satisfactory articial GPx and
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