A selective artificial enzyme inhibitor based on nanoparticle-enzyme interactions and molecular imprinting

Article abstract of DOI:10.1002/adma.201302064

Biological high-performance composites inspire to create new tough, strong, and stiff structural materials. We show a brittle-to-ductile transition in a self-assembled nacre-inspired poly(vinyl alcohol)/nanoclay composite based on a hydration-induced glas

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A Selective Artificial Enzyme Inhibitor Based
on Nanoparticle-Enzyme Interactions and
Molecular Imprinting
Cheng Zheng, Xiao-Long Zhang, Wei Liu, Bo Liu, Huang-Hao Yang,*
Zi-An Lin,* and Guo-Nan Chen
Enzymes, as super-catalysts, play prominent roles in regula-
tion of numerous important biological processes, including
cellular processes and metabolic exchange.[1–3] The discovery
with specific molecular recognition properties of the target mol-
[24–27]
[28–33]
ecules,
including low molecular weight compounds
[34–36]
and biological macromolecules.
Currently, molecular
of efficient enzyme inhibitors as drug candidates is of critical
imprinted polymers (MIPs) used for the recognition and detec-
tion of proteins in biological samples have attracted much
attention. Up to now, some remarkable achievements have
been realized to prepare protein-imprinted polymers, which
relevance to be exploitable for disease treatment.[
4–7]
Nowadays,
[37,38]
small-molecule enzyme inhibitors account for a large scale of
the pharmaceutical market.[ A common problem with con-
ventional small inhibitors, however, is that they not only inhibit
the target enzyme but may also block other enzymes, or act
indiscriminately on healthy and sick cells. For the purpose to
minimize unwanted side effects and quest for selective enzyme
inhibition, this field has been further advanced by the recent
discovery of several selective chemical inhibitors, e.g., octahe-
8,9]
[
39–42]
have wide-ranging applications in separation,
biosen-
[43–45]
[46]
[47]
sors,
enzyme inhibitors.
as enzyme inhibitor by employing a known small-molecule
mimetic enzymes,
protein crystallization
and
[
48,49]
Haupt et al. pioneered MIP microgels
[
48]
enzyme inhibitor as functional monomer.
The inhibition
constant of MIP microgels is almost three orders of magnitude
lower than that of the free small-molecule inhibitor. However, a
known small-molecule enzyme inhibitor for the target enzyme
should be required, which may limit the general applicability of
the strategy. In this work, we combined nanoparticle-enzyme
interactions with molecular imprinting for the first time and
described a novel and general strategy to design selective artifi-
cial enzyme inhibitor.
[
10]
[11]
[12]
dral metal complexes, polymetallic clusters, carboranes
and DNA aptamers.[ Although substantial progress has been
made, the generality of producing such selective chemical
inhibitors remains a problem since the refined X-ray crystal
structures of the target enzymes always should be known. Fur-
thermore, different designs are required for different enzymes.
Also, the preparation processes of such selective chemical
inhibitors are relatively complicated and time-consuming.
Rapid advances in nanotechnology have demonstrated that
nanoparticles are attractive biomaterials due to their unique
13]
Herein, we have chosen the protease α-chymotrypsin (ChT)
as the target enzyme. ChT, whose active site is surrounded by
[
50]
some positive residues, is known as a suitable enzyme for
studying the biomacromolecule surface recognition owning
to its well-characterized geometry and associated enzymatic
activity. In previous report, carbon nanotubes can inhibit the
activity of ChT through electrostatic interactions with the cati-
properties such as simple and low-cost fabrication, size com-
parability and biocompatibility.[
14–17]
In recent years, several
nanoparticles are found to be potent enzyme inhibitors, such
[
18]
[19]
as carbon nanotubes,
graphene oxide,
gold nanoparti-
cles,[
20,21]
dendrimers
[22]
and amphiphilic polymer nanoparti-
onic patch around the enzyme active site. Dopamine (DA)
[51]
cles.[ Unfortunately, such nanoparticle inhibitors may not
be developed as drug candidates due to their poor target-selec-
tivity. Since the enzyme inhibition is mostly attributed to the
electrostatically driven interactions between nanoparticles and
enzymes, nanoparticle inhibitors could not discriminate the
enzymes having the same charge.
23]
is a melanin-like mimic of mussel adhesive proteins. It has
been reported that self-polymerization of DA can produce a
surface-adherent polydopamine (PDA) nanolayer deposited on
[
52]
the surface of multifarious materials at weak alkaline pH.
Nowadays, PDA has been successfully implemented for sur-
[
53,54]
face modification of nanomaterials
imprinting
and surface molecular
[
29,55]
Molecular imprinting technology (MIT) has been accepted
as a cost-effective approach to synthesize artificial antibodies
due to its high stability, hydrophilicity and
biocompatibility. Scheme 1a describes the process flow of the
experiment we conducted to synthesize ChT-selective inhibitor.
As template molecule, ChT was firstly physically adsorbed
on the surface of carboxylic acid-functionalized multiwalled
carbon nanotubes (MWCNT) through electrostatic interactions
in buffered solution to form the MWCNT-ChT complex. Then
an adherent PDA layer was coated on the surface of MWCNT
via polymerization of DA using oxidizing agent under neutral
pH conditions. Finally, MWCNT-MIP was obtained after the
removal of the embedded enzyme molecules.
C. Zheng, X.-L. Zhang, W. Liu, B. Liu, Prof. H.-H. Yang,
Prof. Z.-A. Lin, Prof. G.-N. Chen
The Key Lab of Analysis Technology for Food Safety
of the MOE, College of Chemistry and Chemical
Engineering, Fuzhou University
Fuzhou, 350108, P. R. China
E-mail: hhyang@fio.org.cn; zianlin@fzu.edu.cn
DOI: 10.1002/adma.201302064
Adv. Mater. 2013,
DOI: 10.1002/adma.201302064
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Scheme 1. (a) Schematic illustration of MWCNT-MIP as ChT-selective inhibitor prepared via surface molecular imprinting. (b) Enzymatic hydrolysis
of the chromogenic substrate by free ChT. (c) Inhibition of the adsorbed ChT by MWCNT-MIP.
In this case, MWCNT serve as not only a support material
for the preparation of the MIP layer, but also as functional mon-
omer to participate in binding the active sites of template ChT
molecules and generating the recognition cavities. Due to the
creation of specific binding cavities, the resulting MWCNT-MIP
would achieve the selective recognition as well as the selective
activity inhibition of ChT over other biological macromolecules
layer is successfully polymerized and anchored on the surface
of MWCNT.
To evaluate the imprinting effect of MIPs, the binding iso-
therm is often carried out to get the imprinting factor (IF) and
the specific adsorption capacity. The adsorption isotherms of
MWCNT-MIP and control MWCNT-NIP were investigated by
a batch binding approach with different initial concentrations
of ChT molecules (Figure 2a). The adsorption capacity of ChT
molecules to MWCNT-MIP increased with the increasing of
the initial ChT concentration and came to equilibrium over
(Scheme 1b,c).
The surface morphology of MWCNT and the as-synthesized
MWCNT-MIP were characterized with transmission electron
microscopy (TEM). The average diameter of MWNT was about
−1
0.6 mg mL . Nevertheless, only weak adsorption of ChT mole-
cules to MWCNT-NIP was observed, which may be the result of
nonspecific interactions between the polymer matrix and ChT
molecules. Under this condition, the IF, which is defined as the
ratio of binding capacity of the MIPs with respect to that of the
NIPs, is about 6. It confirms that MWCNT-MIP has high affinity
for ChT molecules. Moreover, a good-linear curve fitting of a
single-site Langmuir-type binding isotherm for MWCNT-MIP is
shown in inset using Scatchard plot equation. According to the
2
0 nm (Figure 1a). And a grey PDA layer with well-defined
configuration and homogeneity was readily observed on the
MWCNT surface as shown in the TEM image of MWCNT-MIP
(Figure 1b). The high-resolution TEM images shown in insets
reveal that the thickness of the PDA layer was about 4 nm.
The X-ray photoelectron spectroscopy (XPS) was employed to
further ascertain the formation of the PDA layer. Figure 1c,d
displays XPS survey spectra of MWCNT before and after
coating a PDA layer. It can be observed that the N1s peak
appeared at about 400.16 eV in the spectrum of MWCNT-MIP
Scatchard analysis, the equilibrium dissociation constant K and
d
the maximum number of binding cavities B
are calculated to
max
−1
(Figure 1d), which is attributed to the nitrogen element of
be 3.65 μM and 134.67 μmol g , respectively. The above data
imply that highly dense recognition cavities are successfully
formed in the imprinted polymer layer by molecular imprinting.
From the binding isotherm of ChT on MWCNT (see Supporting
Information, Figure SI-2), MWCNT exhibited higher adsorption
capacity of ChT molecules than MWCNT-MIP.
amines in the dopamine. While there was no nitrogen signal
detected for MWCNT (Figure 1c). Thermogravimetric analysis
(
TGA) was performed on the basis of the different thermal sta-
bility between MWCNT and PDA layer. From the TGA results
see Supporting Information, Figure SI-1), MWCNT was ther-
(
mally stable up to 600 °C with negligible weight change, yet
MWCNT-MIP showed a significant weight loss below 600 °C
because of the thermal degradation of the PDA layer. The zeta-
potential measurements indicated that MWCNT exhibited a
zeta-potential of about –60.3 mV at pH 7.4. After imprinting,
the zeta-potential of MWCNT-MIP was increased to –40.0 mV.
The results above support that a uniform and nanoscale PDA
The binding kinetics given in Figure 2b describes the time-
dependent evolution of the ChT amount bound by MWCNT-
MIP and MWCNT-NIP. MWCNT-MIP reached the maximum
adsorption capacity within 1 h, revealing a rapid adsorption
of ChT molecules into MWCNT-MIP. Given the fact that the
thickness of the PDA layer is comparable to the hydrodynamic
[56,57]
radius of ChT (2.5–2.8 nm),
template ChT molecules are
2
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(
c)2
(d)2.0x10
1.5x10
5
5
5
4
5
5
5
4
.0x10
.5x10
.0x10
.0x10
C1s
1
1
5
C1s
O1s
1.0x10
O1s
N1s
5.0x10
0
.0
6
0.0
600
00
500
400
300
200
100
0
500
400
300
200
100
0
Binding Energy, eV
Binding Energy, eV
Figure 1. TEM images of MWCNT (a) and MWCNT-MIP (b), respectively. The insets show the high-resolution TEM images, which indicate that the MIP
layer was uniform and nanoscale. XPS patterns of MWCNT (c) and MWCNT-MIP (d), respectively. A distinct N1s peak was observed in the spectrum
of MWCNT-MIP.
presumed to be not fully encapsulated in the PDA layer. So, the
ease of template removal as well as rebinding can be improved.
It is also implied that there should be monolayer coverage of
the recognition cavities on the MWCNT surface. Furthermore,
MWCNT participates in binding the active sites of template
ChT molecules in the conduction of the molecular recogni-
tion cavities. So in the rebinding process, the active sites of the
adsorbed ChT molecules are presumed to direct towards the
MWCNT surface. As a result, the activity of the adsorbed ChT
molecules would be inhibited.
(
a) 3.5
(b) 3.5
MWCNT-MIP
MWCNT-NIP
MWCNT-MIP
MWCNT-NIP
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3.0
25
2.5
2.0
1.5
1.0
0.5
0.0
2
1
1
0
5
0
5
0
4
0
60
80
100
120
140
-
1
B, µmol g
0
.0
0.2
0.4
0.6
0.8
1.0
0
20
40
60
80 100 120 140 160
-1
Time, min
ChT, mg mL
−
1
Figure 2. (a) Binding isotherms of ChT (0.1–1.0 mg mL ) on MWCNT-MIP ( ) and MWCNT-NIP (᭹). The inset shows Scatchard analysis of the binding
᭿
−
1
capacity of ChT to MWCNT-MIP. (b) Binding kinetics of ChT (0.4 mg mL ) on MWCNT-MIP ( ) and MWCNT-NIP (᭹).
᭿
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(b)100
(
a)
1
0
.5
ChT Activity
-1
ug mL
MWCNT-MIP
MWCNT-NIP
0
.0
.8
.6
.4
.2
.0
0.4
-1
50 ug mL
80
1
0
.3
.2
.1
.0
0
0
0
0
0
0
60
40
20
0
0
0
0
80 160 240 320 400 480
Time, s
0
20
40
60
80 100 120 140 160
20
50
80
100
150
-1
-1
MWCNT, µg mL
MWCNT-MIP / MWCNT-NIP, µg mL
−
1
Figure 3. (a) Activity of ChT (500 μg mL ) plotted as a function of MWCNT concentration. The inset shows UV-vis absorbance change of the system
−
1
at 405 nm with the time after incubation with different concentrations of MWCNT (0–150 μg mL ). (b) Degrees of inhibition of the ChT activity after
−
1
−1
incubating ChT (500 μg mL ) with different concentrations of MWCNT-MIP and MWCNT-NIP (0–150 μg mL ). The activities were normalized to that
of free ChT. The data shown here represented the means and standard deviations of three independent experiments.
In general, ChT hydrolyses polypeptides at the carboxyl-
side of a Tyr, Trp or Phe residue. The ChT activity is com-
monly detected with chromogenic substrate, N-succinyl-L-
phenylalanine-p-nitroanilide (SPNA), to provide color readout.
The enzyme-inhibition experiment was performed to evaluate
the inhibition efficiency of MWCNT by the ChT-catalyzed
hydrolysis of SPNA after incubating ChT with various concen-
trations of MWCNT. As shown in Figure 3a, the rate of enzy-
matic hydrolysis decreased upon addition of MWCNT, and the
strongest inhibitory potency was around 92% ChT activity sup-
Therefore, inhibitory potency of MWCNT-NIP is low. In the
meantime, MWCNT-MIP has higher inhibitory potency in
[48]
comparison with the reported MIP microgels. We attribute
the higher inhibitory potency of MWCNT-MIP to two fea-
tures. First, MWCNT-MIP has good site accessibility towards
the target enzyme via surface molecular imprinting. Second,
nanoscale MWCNT-MIP has extremely high surface-to-volume
ratio, which provides larger enzyme binding capacity and
higher inhibition efficiency.
High target-selectivity has been regarded as one of the most
fascinating properties of MIPs. Four non-template proteins
with the different isoelectric points (pI) and molecular weights
(Mw), namely, trypsin, cytochrome c (Cyt), bovine serum
albumin (BSA), myoglobin (Mb), were subjected as the con-
trols to study the binding selectivity of MWCNT-MIP. It is seen
from Figure 4a that MWCNT-MIP exhibited a high adsorp-
tion capacity of ChT molecules. However, the binding capacity
of other proteins to MWCNT-MIP was very low. Based on the
above results, it is demonstrated that MWCNT-MIP has consid-
erably high selectivity towards ChT molecules, which involves
multiple weak interactions provided by functional monomers
and stereo-shape complementarities.
−1
pression at a MWCNT concentration of 150 μg mL .
In the case of MWCNT-MIP (Figure 3b), the inhibition
tendency was similar to that of MWCNT. The ChT activity
was profoundly affected by MWCNT-MIP with a loss of 85%
−1
activity at concentrations up to 150 μg mL . On the other
hand, the activity of ChT only decreased 13% when the dosage
−1
of MWCNT-NIP increased to 150 μg mL . It is apparent that
MWCNT-MIP provides much higher inhibition efficiency than
MWCNT-NIP at equivalent dose. It is attributed to the recog-
nition cavities within MIP layer created by template ChT mol-
ecules. On the contrary, NIP layer without recognition cavities
could prevent ChT molecules to attach to the MWCNT surface.
(
a) 3.5
(b)
MWCNT-MIP
MWCNT-NIP
MWCNT
MWCNT-MIP
1
.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.8
0.6
0.4
0.2
0.0
0
5
10
15
20
25
30
ChT trypsin Cyt
BSA
Mb
-
1
MWCNT / MWCNT-MIP, µg mL
Figure 4. (a) Selective adsorption of ChT (Mw = 25,000, pI = 9.1), compared with trypsin (Mw = 23 800, pI = 10.5), Cyt (Mw = 12 400, pI = 10.2), BSA
−
1
(
Mw = 66 000, pI = 4.8), Mb (Mw = 17 600, pI = 7.1), on MWCNT-MIP and MWCNT-NIP. (b) Activity of trypsin (15 μg mL ) plotted as a function of
−1
MWCNT and MWCNT-MIP concentrations (0–30 μg mL ). The activities were normalized to that of free trypsin. The above data shown here repre-
sented the means and standard deviations of three independent experiments.
4
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After the selective molecular recognition property of
MWCNT-MIP was confirmed, its selective inhibitory response
for ChT was further investigated. Trypsin was selected as a
control enzyme. Trypsin is also a serine protease with sim-
ilar molecular weight and isoelectric point compared to ChT.
Team in University (No. IRT1116) and the National Science Foundation
of Fujian Province (No. 2010J06003).
Received: May 7, 2013
Revised: July 11, 2013
Published online:
97
75
62
96
Some basic residues, for example, Lys , Lys , Arg and Arg ,
distribute over the trypsin surface.[ Obviously, MWCNT could
deactivate trypsin (Figure 4b). In marked contrast, MWCNT-
MIP only showed slight inhibitory activity towards trypsin.
This observation can be explained by the fact that the recogni-
tion cavities within MIP layer do not fit the size and shape of
trypsin. The inhibition studies above expressly verify that the
selective feature of the nanoparticle inhibitor is significantly
improved when combined with molecular imprinting.
58]
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