Control of protein-binding kinetics on synthetic polymer nanoparticles by tuning flexibility and inducing conformation changes of polymer chains

Article abstract of DOI:10.1021/ja306053s

Although a number of procedures to create synthetic polymer nanoparticles (NPs) with an intrinsic affinity to target biomacromolecules have been published, little has been reported on strategies to control the binding kinetics of target recognition. Here,

Full text of DOI:10.1021/ja306053s

Communication
pubs.acs.org/JACS
Control of Protein-Binding Kinetics on Synthetic Polymer
Nanoparticles by Tuning Flexibility and Inducing Conformation
Changes of Polymer Chains
Yu Hoshino,* Masahiko Nakamoto, and Yoshiko Miura*
Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
*
S Supporting Information
22
sites of the protein. Large conformation changes of DNA
polymerase accelerate the binding rate and decelerate the
dissociation rate of the DNA capture process, resulting in
ABSTRACT: Although a number of procedures to create
synthetic polymer nanoparticles (NPs) with an intrinsic
affinity to target biomacromolecules have been published,
little has been reported on strategies to control the binding
kinetics of target recognition. Here, we report an enzyme-
mimic strategy to control binding/dissociation rate
constants of NPs, which bind proteins through multipoint
interactions, by taking advantage of the temperature-
responsive coil−globule phase transition of poly-N-
isopropylacrylamide (PNIPAm)-based NPs. PNIPAm
NPs with a “flexible” random-coil conformation had a
faster binding rate than NPs with a “rigid” globule
conformation; however, the dissociation rate constant
remained unchanged, resulting in stronger affinity. The
dissociation rate of the “flexible” NPs was decelerated by
the “induced-fit”-type conformation change of polymers
around the coil−globule phase transition temperature,
resulting in the formation of the most stable NP−protein
complexes. These results provide a guide for designing
plastic antibodies with tailor-made binding kinetics and
equilibrium constants.
2
0,21
formation of a stable DNA−protein complex.
In this study, we mimic the flexibility and conformational
change of enzymes to control the binding kinetics of target
molecules on synthetic polymer NPs. To induce the flexibility
and conformation change of the NPs, we took advantage of the
temperature-responsive coil−globule phase transition of
PNIPAm-based hydrogel NPs. PNIPAm NPs undergo a coil−
globule phase transition at the lowest critical solution
23
temperature (LCST, approximately 37 °C). At 25 °C,
PNIPAm chains are in the random-coil conformations, and
thus, the PNIPAm NPs are swollen, though they are in the
globule conformation and collapsed above their transition
temperature as a result of an entropy-driven dissociation of
24
1
water molecules. H NMR revealed that the isopropyl group
has a large mobility in the swollen state; however, the mobility
25
decreases substantially upon collapse of the gel. Time-
resolved fluorescence anisotropy measurements revealed that a
marked reduction in the segmental mobility of the PNIPAm
26
chains also occurs at the onset of the LCST. Therefore,
flexibility of PNIPAm chains can be tuned from the “flexible”
swollen phase with greater segment and side chain motions to
the “rigid” collapse phase depending on temperature. The
LCST can be further tuned by incorporating N-tert-
butylacrylamide (TBAm) into NPs.
It has been reported that diffusion of small molecules in the
ynthetic polymer nanoparticles that recognize, capture,
S
and/or release specific biomacromolecules are of significant
1
−3
interest as drug carriers, affinity ligands, and antidotes.
23
Recently, nanoparticles (NPs) that recognize target proteins/
peptides via multipoint interactions have been achieved by
optimizing combinations and populations of functional
PNIPAm hydrogels can be reversibly switched on and off by
27,28
4
−8
9,10
collapse and swell phase transition.
Diffusion rate of the
groups
and/or imprinting binding-sites
in poly-N-
molecules in PNIPAm can be further tuned by hydrophobic
isopropylacrylamide (PNIPAm)-based hydrogel NPs. Some
NPs have shown the ability to recognize, neutralize, and/or
29,30
interaction.
Tanaka and co-workers reported that with
1
0,11
cationic functional groups, PNIPAm gels capture polyanionic
release target molecules, even in living animals.
However,
31
dyes through multipoint interactions. The interaction can be
further switched on/off by increasing/decreasing volume
density of the functional group with the collapse/swell phase
the activity of the particles in vivo was significantly lower than
11
that expected based on results from in vitro experiments,
since undesired events such as creation of protein coronas on
31
transition of the PNIPAm gels. Shea and co-workers have
NPs and metabolism of materials compete with the designed
3
7
1
2−15
extended the strategy to protein and peptide targets using
functionalized PNIPAm NPs. However, effects of flexibility and
conformation changes of PNIPAm NPs on the binding/
dissociation rate of the multipoint-protein-binding process have
not been revealed. To investigate contribution of the flexibility
and conformation change of NPs on the protein-binding
function.
To utilize materials in nonequilibrium living
systems, binding kinetics as well as the equilibrium of the
1
1,13,16
target-binding process must be carefully engineered.
17−19
It has been reported that the flexibility
and conforma-
of enzymes play a crucial role on the
2
0,21
tional changes
determination of the binding and dissociation rates of the target
binding process. For instance, the target binding/dissociation
kinetics of dihydrofolate reductase are determined by motion of
a flexible loop that covers the substrate- and coenzyme-binding
Received: June 21, 2012
Published: September 4, 2012
©
2012 American Chemical Society
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Journal of the American Chemical Society
Communication
process, it is important to maintain the volume density of
functional groups, which contact (interact) with target protein,
in NPs, because dissociation (equilibrium) constants increase
exponentially with the volume density of the functional group
31
11
in the bulk gels and NPs, then effects of flexibility and
conformation changes of NPs would be hidden in the effects of
density of functional groups. It is also necessary to compare
binding/dissociation rate of NPs with different flexibility at the
same temperature, because diffusion constants, which are
associated with binding/dissociation constants, increase ex-
ponentially with the temperature (the Arrhenius equation). In
this study, we carefully prepared NPs having various phase
(flexibility) at 25 °C with identical volume-density of functional
group and quantified binding/dissociation rate constants on
each NPs at the same temperature (25 °C).
Figure 1. (a) Hydrodynamic diameter of ManNPs synthesized with
0
% (NP1; open circle), 40% (NP2; black triangle), and 20% (NP3;
As a model of a target molecule that would be recognized by
PNIPAm NPs, concanavalin A (Con A), a 104 kDa α-D-
mannose-binding protein was selected. It is a homotetrameric
protein having 4 mannose-binding sites on the corners of its
green square) TBAm as a function of temperature. (b) Schematic of
the QCM experiments. ManNPs were immobilized on the Au surface
of the QCM sensor through Con A-mannose interaction (left). Con A
binding on ManNPs was monitored on the surface of the sensor
following introduction of Con A (right). (c) An AFM image of
ManNPs (40% TBAm, 2% BIS, 5% AAc, 0.11% Man) immobilized on
a Au substrate through Con A−mannose interaction. A height profile
of the cross section (light blue line) is shown inset.
32
tetrahedral structure, and each side is 10 nm long. Although
the monovalent mannose-Con A interaction is relatively weak
−
3
−4
(
K 10 −10 M), it can be amplified by multivalency; by
d
clustering a number of mannose units on a polymer chain, a
synthetic polymer that recognizes Con A with much stronger
affinity can be achieved (K 10 −10 M). Thus, Con A has
been widely used as a model target molecule for the study of
−5
−8
33
d
and swell ratio of NP3 were almost the same as NP2 at 25 °C,
NP3 was apparently not in the fully swollen state at 25 °C
because NP3 gradually shrunk around 25 °C (between 20 and
synthetic polymer architectures that recognize specific bio-
34−38
macromolecules through multipoint interactions.
p-Acrylamidophenyl-α-D-mannopyranoside (Man) was syn-
3
5 °C) (Figure 1a). To represent the state of NP3 at 25 °C, we
defined the state as “transition state”. To improve colloidal
stability and to control the swell ratios of the NPs, 5 mol % of
acrylic acid and 10 mol % of BIS (N,N′-methylenebisacryla-
mide) were incorporated into all NPs. All NPs did not form
aggregates, and did not show significant size and LCST changes
in water for at least one year at room temperature (Figure S3).
The binding rate of Con A to the NPs was monitored and
analyzed using a 27 MHz quartz crystal microbalance (QCM)
36,39
thesized as described (Supporting Information (SI))
and
copolymerized into PNIPAm NPs to prepare NPs that
recognize Con A through multipoint interactions (Scheme 1).
Scheme 1. Preparation of ManNPs
35,40
(
Figure 1b).
Prior to studying the binding kinetics, binding
specificity between the NPs and Con A was confirmed by
several experiments. First, when NPs that had Man (NP1−3)
were injected into the QCM cells on which Con A had been
immobilized, significant interactions were observed. However,
no interaction was observed when the NPs without Man were
injected (Figure S4). Second, when Con A was injected into the
cells on which only Con A had been immobilized, no
interactions were observed (Figure S4); in contrast, when
Con A was injected into the cells on which ManNPs (NP1−3)
had been immobilized on the surface of Con A, significant
interactions were observed (Figure 2a). Furthermore, it was
confirmed by atomic force microscopy that immobilization of
NPs on the surface of gold (Au (111) on mica, Phasis) was not
greater than a single layer (Figure 1c).
To reveal the influence of flexibility and conformation changes
of NPs on the binding kinetics of Con A at 25 °C, NPs with
LCST below (NP1, <10 °C), above (NP2, 37 °C), and around
(
NP3) 25 °C were prepared by tuning the feed ratio of TBAm
(
Figure 1a). The volume density of Man in NP1−NP3 was
Figure 2a shows typical frequency changes as a function of
time for the ManNP-immobilized QCM in 10 mM phosphate
buffer (pH 7.4, 137 mM NaCl, 2.68 mM KCl, 1.8 mM CaCl2,
3
maintained at 1.0 ± 0.1 molecules/(10 nm) at 25 °C by
controlling the feed ratio of Man, depending on the swell ratio
of each type of NPs (Table S1). It was expected that the Man
and 0.49 mM MgCl ) responding to the addition of Con A.
2
3
density of 1.0 ± 0.1 molecules/(10 nm) allowed multipoint
When Con A was injected into the cell (5−240 nM), the
frequency decreased (the mass of Con A on the surface
increased) gradually for 10−30 min because of the binding of
Con A on the ManNPs. Both the binding rate (relaxation time
of the binding process) and the binding amount of Con A
depended on the concentration of Con A (Figure 2a).
interactions between several Man units on the NPs and a Con
A molecule, depending on the flexibility of the polymer chains
of the ManNPs. Incorporation of Man into each NP was
1
quantified by H NMR (Figures S1 and S2) and the swell ratios
were quantified by dynamic light scattering (DLS). As we
designed, NP1 and NP2 were in the swollen and collapsed
states, respectively, at 25 °C (Figure 1a). Although diameter
The QCM frequency changes in the Con A binding
processes were fit very well by the single exponential function
1
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Journal of the American Chemical Society
Communication
Figure 2. (a) Time courses of frequency changes of NP1-immobilized QCM, responding to the addition of 5 (red), 10 (green), 20 (sky blue), 30
orange), 60 (black), and 240 (blue) nM Con A. Results of duplicated experiments (thick lines) and fit curves (thin lines) are overwritten. (b) Plots
(
of binding relaxation time (τ) on swollen (NP1, open circle), collapsed (NP2, black triangle), and transition state (NP3, green square) NPs as a
function of Con A concentration. (c) Volume shrinkage of swollen (NP1, gray), collapsed (NP2, black), and transition state (NP3, green) NPs that
were induced by Con A binding.
a
Table 1. Composition, Phase, Man Density, and Rate/Equilibrium Constants of PNIPAm NPs
Density of Man [molecule (10 nm)−3] kon 10 M−1·s−1 koff 10−3 s−1 Kd nM
3
NPs TBAm feed [mol %] Man feed [mol %]
State at 25 °C
1
2
3
0
40
20
1.5
Swollen phase
Collapsed phase
Transition
1.1
0.9
0.9
18
8
1.4
1.2
0.8
78
150
38
0.11
1.5
21
a
10 mol % BIS and 5 mol % AAc were fed in all NPs.
24−26
(eq 1, Figure 2a), indicating that the binding event observed in
conformation in the collapsed gels.
It is suggested that
this experiment can be approximated as interaction between
Con A and the binding sites on the NPs, each of which has an
equal affinity, although the binding site in each of the NPs
the flexibility of the polymer chains in the swollen NPs allowed
mannose to map onto mannose-binding sites on the surface of
Con A resulting in faster binding and stabilization of the NP-
Con A complexes.
6
cannot be homogeneous (SI).
The transition state NPs showed lower dissociation rate
ΔF = ΔF (1 − e−
t/τ
)
(1)
−3 −1
−3
∞
constant (0.8 × 10 s ) than the swollen NPs (1.4 × 10
−
1
s ), although the binding rate constants of each of the NPs
were almost same. Therefore, the transition state NPs showed
The reciprocal of the binding relaxation time (τ) for each of
the binding processes was plotted against Con A concentration
Figure 2b), showing linear correlations between concentration
and τ for each type of NPs. These results indicate that the
binding process can be approximated as the Langmuir-type
binding process given in eq 2 (SI). Apparent binding/
an even stronger affinity to Con A (K = 38 nM) than the
(
d
−1
swollen NPs (K = 78 nM).
d
To investigate the contribution of conformation changes of
polymers on the binding kinetics, volume shrinkage of each of
the NPs, which were induced by Con A binding, were
estimated by comparing the size of NPs before and after
addition of Con A by DLS in 10 mM HEPES buffer (pH 7.4,
dissociation rate constants (kon and k ) for each type of NPs
off
were given as the slopes and y-intercepts of the linear
3
5,40
correlations (eq 3, SI):
1
.8 mM CaCl , and 0.49 mM MgCl ). Interestingly, the volume
2
2
ConA + NP ⇄ ConA·NP
τ− = k [ConA] + k
(2)
(3)
of the transition state NPs shrunk by 20−40% after addition of
a small amount of Con A, depending the on concentration of
Con A added (60−120 nM). In contrast, the swollen phase
NPs showed a significantly lower degree of size shrinkage (10−
1
on
off
The obtained rate constant k , k , and dissociation
on
off
1
5%) than the transition state NPs (Figure 2c). Collapsed NPs
equilibrium constant K (=k /k ) values are summarized in
d
off on
did not show any volume shrinkage (Figure 2c). These results
indicate that polymer density (steric hindrance) around the
binding sites on the transition state NPs after Con A binding
was much greater than in the prebinding structure owing to
volume shrinkage, resulting in deceleration of the dissociation
Table 1.
Dissociation constants for NP1−3 were in the submicromo-
lar range (78, 150, and 38 nM, respectively, Table 1). This
number is more than 3 orders of magnitude smaller than the
−
3
−4
reported monovalent Man−Con A interaction (K 10 −10
d
41
3
3
rate. This is comparable to the ‘‘induced-fit’’ between
M), indicating that Con A binding on NP1−3 observed in
those experiments are all multivalent interactions between more
than 2 Man units on a particle and one Con A.
4
2,43
enzymes or gel catalysts and substrates.
From these results, we conclude that binding rates and
equilibrium constants can be tuned by the choice of phase of
NPs. Swollen phase NPs showed a faster binding rate and
stronger affinity than collapsed phase NPs since swollen NPs
consist of random-coil-phase polymer chains which are more
flexible than the globule-phase chains in collapsed NPs. NPs
around the coil−globule phase transition temperature, which is
defined as transition state NPs, showed an even stronger affinity
than the swollen NPs since bound proteins induced greater
conformation change of NPs, resulting in shrinkage of NPs and
Although the dissociation rate constants (k ) of swollen (1.4
10 s ) and collapsed (1.2 × 10 s ) NPs were almost
equal, the binding rate constant (k ) for the swollen NPs (18
off
−3
−1
−3 −1
×
on
3
−1 −1
×
1
10 M s ) was larger than that of the collapsed NPs (8 ×
3
−1 −1
0 M s ). As a result, the dissociation constant (K ) of the
d
swollen NPs (78 nM) was smaller than that of the collapsed
NPs (150 nM). Polymer chains in the swollen gels are in the
flexible random-coil conformation with larger segment mobility,
whereas the polymer chains are in the rigid globule
1
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Journal of the American Chemical Society
Communication
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures and supporting data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
yhoshino@chem-eng.kyushu-u.ac.jp; miuray@chem-eng.
kyushu-u.ac.jp
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(
ACKNOWLEDGMENTS
Financial support from MEXT (23111716 and 20106003) and
JSPS (23750193) is greatly appreciated.
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