Design of multi-functional linear polymers that capture and neutralize a toxic peptide: A comparison with cross-linked nanoparticles

Article abstract of DOI:10.1039/c4tb01967a

In this paper, a library of multi-functional linear poly-N-isopropylacrylamide (pNIPAm) polymers having a range of molecular weights and functional groups were synthesized and their interaction with the hemolytic peptide, melittin, was examined. The linear pNIPAm (LPs) containing both tert-butyl group and carboxylic acids bound with the peptide by a combination of hydrophobic and electrostatic interactions and neutralized its toxicity. The melittin binding capacity and affinity of each LP was quantified and further compared with cross-linked multi-functional nanogel particles (NPs) having same combination of functional groups. The binding capacity of the LPs (weight of captured melittin/weight of LP) was independent of their molecular weight and was three times higher than that of previously reported NPs. The binding constant depended on the molecular weight of the LPs, showing the highest value of 1.1 × 10⁸ (M^-1) for a ～1000 mer linear polymer with 40% tert-butyl group and 20% carboxylic acid. Comparison of the interactions of the LPs and NPs suggested the importance of the flexibility of the polymer chain in order to achieve high binding capacity and affinity. This journal is

Full text of DOI:10.1039/c4tb01967a

Journal of
Materials Chemistry B
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PAPER
Design of multi-functional linear polymers that
capture and neutralize a toxic peptide: a
comparison with cross-linked nanoparticles†
Cite this: J. Mater. Chem. B, 2015, 3,
706
1
a
a
a
b
c
Yusuke Wada, Haejoo Lee, Yu Hoshino,* Shunsuke Kotani, Kenneth J. Shea
a
and Yoshiko Miura
In this paper, a library of multi-functional linear poly-N-isopropylacrylamide (pNIPAm) polymers having a
range of molecular weights and functional groups were synthesized and their interaction with the
hemolytic peptide, melittin, was examined. The linear pNIPAm (LPs) containing both tert-butyl group and
carboxylic acids bound with the peptide by a combination of hydrophobic and electrostatic interactions
and neutralized its toxicity. The melittin binding capacity and affinity of each LP was quantified and
further compared with cross-linked multi-functional nanogel particles (NPs) having same combination of
functional groups. The binding capacity of the LPs (weight of captured melittin/weight of LP) was
independent of their molecular weight and was three times higher than that of previously reported NPs.
The binding constant depended on the molecular weight of the LPs, showing the highest value of 1.1 ꢀ
Received 27th November 2014
Accepted 15th January 2015
8
ꢁ1
10
(M ) for a ꢂ1000 mer linear polymer with 40% tert-butyl group and 20% carboxylic acid.
DOI: 10.1039/c4tb01967a
Comparison of the interactions of the LPs and NPs suggested the importance of the flexibility of the
polymer chain in order to achieve high binding capacity and affinity.
www.rsc.org/MaterialsB
It has been reported that pNIPAm NPs, randomly copoly-
merized with a 2–10 mol% cross-linker and combinations of
1
. Introduction
The strong and specic interactions between bio- functional monomers were capable of binding peptides and
macromolecules such as proteins and peptides are achieved proteins via a combination of hydrogen bonding, hydrophobic,
7
through a combination of van der Waals, electrostatic and aromatic and/or electrostatic interactions. Dawson, Linse and
hydrophobic interactions on complementary three-dimensional their co-workers have shown that NPs with optimized size and
1
binding surfaces. As such, protein-mimicking materials with hydrophobicity interact with proteins and peptides and accel-
8
affinity for specic peptides or proteins might be achieved erated or inhibit nucleation of brillation. The affinity to the
through the incorporation of an optimized combination and/or target can be enhanced by optimization of the volume density of
9
–11
12
the ratio of functional groups on the surface of the nano- functional groups in the NPs,
material. For example, gold nanoparticles and dendrimers
molecular imprinting,
2
3
4
13
14
15
affinity purication and tuning the exibility and density of
that express the appropriate combination and/or density of polymer chains in NPs. NPs designed in this manner have been
charged and/or hydrophobic functional groups on their surface shown to neutralize target toxins in vitro and in vivo, and have
10,16
are capable of recognizing proteins. Schrader and co-workers been termed “plastic antibodies”.
These polymers could
have designed molecular ligands that interact strongly with potentially be used as stable and inexpensive materials for the
5
17,18
arginines and lysins. Then, displayed the molecular ligands on purication of proteins and protein complexes.
However, in
the side-chain of linear copolymers and dendrimers together the design of multifunctional polymers, the inuence of the
with hydrophobic, hydrophilic and charged functional group to molecular weight and higher-order structure of the polymer
recognize target proteins thru combination of electrostatic, chains on their ability to recognize and neutralize biomolecules
6
hydrophobic and hydrogen bonding interaction.
is not clear. Physical and chemical crosslinks inside the gel
particles may limit the access of target molecules to the internal
portion of the NPs or cause a reduction of the exibility of the
a
Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishi-ku, polymer chain, inhibiting the formation of stable target–poly-
Fukuoka 819-0395, Japan. E-mail: yhoshino@chem-eng.kyushu-u.ac.jp
mer complex.
b
Priority Organization for Innovation and Excellence, Kumamoto University,
In this study, a library of multifunctional linear pNIPAm
Kumamoto 862-0973, Japan
c
(LPs) was synthesized and the inuence of the combinations
Department of Chemistry, University of California, Irvine, CA 92697, USA
Electronic supplementary information (ESI) available: Binding isotherms of LPs
to melittin immobilized QCM. See DOI: 10.1039/c4tb01967a
and incorporation ratio of functional groups and molecular
weight on the interaction with a model toxic peptide was
†
1706 | J. Mater. Chem. B, 2015, 3, 1706–1711
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LP300). NIPAm was used as the principle monomer and TBAm
and AAc were used as functional monomers with a hydrophobic
(tert-butyl) and a negatively charged (carboxylate anion) side
chain. A total of 22 types of multifunctional polymers were
prepared by changing the feed ratio of TBAm and AAc from 0–
1
4
0% and 0–20%, respectively (Table 1). H NMR conrmed that
in a 90% or higher conversion of all LPs and an incorporation
ratio in accordance with the feed was achieved. The PDI,
apparent M and M were measured by GPC using polystyrene
n w
as standard. The GPC results showed that all LPs have mono-
modal size distribution. The PDIs were approximately 1.50–2.25
for LP1000 and 1.38–1.82 for LP300, indicating that polymeriza-
Scheme 1 (a) Crystal structure and amino acid sequence of melittin.
Hydrophobic, and positively charged, amino acids are printed in green,
and red respectively. The PDB ID is 2MLT. (b) Schematic of the binding tion process was not perfectly controlled comparing with the
between melittin and the ideal LP. Hydrophobic, and negatively
charged functional groups are printed in green, and blue respectively.
19
n w
previous report. It was difficult to obtain M and M that
match up precisely with the designed value. Because there is
signicant interaction between carboxylic acids on LPs and
(
c) Preparation of multifunctional LPs.
20
stationary phase in the GPC column. However, all NPs poly-
merized with small population of AAc showed that the addition
of chain transfer agent allowed for the synthesis of LPs with
controlled molecular weights. Although, PDI, Mn and Mw of
some LPs were not perfectly controlled as designed, we decided
to use the library for the further study, since the LPs in the
library have well dened properties enough to compare melittin
neutralization activity with NPs and to study the inuence of the
combinations and incorporation ratio of functional groups and
molecular weight on the interaction with a model toxic peptide.
analyzed. Melittin, a peptide composed of 26 amino acids, was
selected as the target toxic peptide. Melittin is a hemolytic toxin
from bee venom and has six positively charged amino acids and
a large number of hydrophobic amino acids (Scheme 1a). It has
been reported that melittin is captured by cross-linked pNIPAm
NPs, which are copolymerized with negatively-charged acrylic
acid (AAc) and hydrophobic N-tert-butylacrylamide (TBAm)
through combination of electrostatic and hydrophobic inter-
7,10
actions (Scheme 1b). Therefore, in this study, AAc and TBAm
were selected as the functional monomers. LPs with two
different molecular weights (300 mer and 1000 mer; LP300 and 2.2. LP neutralization and binding capacity of melittin
LP1000 respectively) were synthesized by UV-initiated reversible
addition fragmentation transfer polymerization in the presence
of chain transfer agent (CTA) as reported. The binding capacity
The neutralization of hemolytic toxicity of melittin by LPs was
7
tested by the red blood cell lyses test as previously reported.
ꢁ1
First, 100 mg mL of polymer was mixed with 1.8 mM of melittin
and affinity with melittin was analyzed by peptide neutraliza-
and red blood cells, and the mixture was incubated for 30 min.
LPs with only AAc or TBAm were unable to neutralize the
hemolytic activity of melittin substantially (inhibition ratio <
7,10
tion assay and quartz crystal microbalance. The results were
7,10
further compared with the previously reported results of NPs.
Since little have been reported about the inuence of the
molecular weight, higher-order structure and exibility of
synthetic polymers on their ability to recognize targets, this
study may be useful in the future design of synthetic polymers
capable of strongly binding to target biomacromolecules.
40%) (Fig. 1a and b). On the other hand, most LP1000 and LP300,
which contained both AAc and TBAm, showed a high inhibition
ratio (>60%), indicating that melittin was captured by
LP through combination of electrostatic and hydrophobic
interaction regardless of their molecular weight (300 mer or
1000 mer).
Binding capacity of melittin to LPs was calculated from the
2
. Results and discussion
relationship between concentration of LPs and inhibition ratio
2.1. Synthesis of a linear polymer library
7,10
of red blood cell lyses as reported (Fig. 1c and d).
LPs con-
It has been reported that poly-AAc can be synthesized in taining 40% TBAm and 5% or higher of AAc showed the highest
ꢁ1
aqueous media by UV- or g-ray initiated living radical poly- binding capacity of 1.5 g g , and the binding capacity did not
merization by the reversible addition fragmentation chain increase when the incorporation of AAc was increased from 5%
19
transfer (RAFT) mechanism. In this study, we modied the to 20% (Fig. 1c and d and 2). A comparison between LP300 and
procedure to synthetize a library of multifunctional polymer, LP1000 showed that the binding capacity did not depend on the
since the TBAm and chain transfer agent (benzylsulfanylth- molecular weight of the polymers (Fig. 1c and d and 2a).
iocarbonylsulfanyl propionic acid; BPATT) was not soluble in
It has been reported that cross-linked NPs that were poly-
water in the absence of AAc. We used methanol was as solvent to merized with the same combination of functional monomers as
dissolve TBAm and BPATT, and polymerization reaction was LPs captures melittin thru combination of electrostatic and
ꢃ
19
7,10
carried out at 70 C under UV-irradiation (Scheme 1). LPs with hydrophobic interaction. The binding capacity of NPs, which
two different molecular weights were prepared by tuning the hydrodynamic diameter was 50–90 nm, was about 3 times
10,18
stoichiometric ratio of the total monomer concentration to the smaller than LPs (Fig. 2a).
This suggests the presence of a
chain transfer agent to be 1000 and 300, respectively (LP1000
,
domain inside the NPs, which could not bind to melittin.
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Table 1 Feed ratio of functional monomers, conversion of polymerization reaction, incorporated ratio of functional monomers, M
w
, M
n
and PDI
of LPs
Feed ratio
mol%)
Incorporated ratio
(mol%)
(
LPs
Feed ratio [monomer]/[CTA]
TBAm
AAc
Conversion (%)
TBAm
AAc
PDI
LP1000-1
LP1000-2
LP1000-3
LP1000-4
LP1000-5
LP1000-6
LP1000-7
LP1000-8
LP1000-9
LP1000-10
LP1000-11
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
0
0
5
20
0
1
5
20
0
1
5
10
20
97
98
93
96
98
96
96
99
98
95
95
0
0
5
21
0
1
5
20
0
1
5
10
20
1.85
1.50
1.53
1.85
1.77
1.54
1.50
1.65
2.25
1.71
1.70
20
20
20
20
40
40
40
40
40
20
20
20
20
40
40
40
40
40
LP300-1
LP300-2
LP300-3
LP300-4
LP300-5
LP300-6
LP300-7
LP300-8
LP300-9
LP300-10
LP300-11
300
300
300
300
300
300
300
300
300
300
300
0
0
5
20
0
1
5
20
0
1
5
10
20
98
93
97
99
97
93
99
98
98
95
83
0
0
5
20
0
1
5
20
0
1
5
10
22
1.50
1.68
1.38
1.54
1.67
1.60
1.38
1.48
1.59
1.68
1.82
20
20
20
20
40
40
40
40
40
20
20
20
20
40
40
40
40
38
Fig. 2 Effect of the amount of AAc on the melittin-binding capacity of
10
ꢁ1
NPs (green), LP300 (blue), and LP1000 (red) containing 40% TBAm.
Fig. 1 Hemolytic activity inhibition ratio with 100 mg mL LP1000 (a)
and LP300 (b). Gray cones indicate almost complete neutralization of
melittin. Melittin-binding capacity of LP1000 (c) and LP300 (d). Binding
capacity of LPs shown as black parallelogram was too small to be
calculated.
water) for both monomers and polymers. It also suggested steric
hindrance was eliminated by using linear polymers, thus
allowing the binding of melittin on most parts of the polymer
chain.
The binding capacity of 1.5 g g suggests that one molecule
of melittin was captured by 20-units of monomer on a LPs
Chemical crosslinks in the gel particles formed by crosslinking
ꢁ
1
21
monomers and intra particle radical chain transfer and/or
physical crosslinks formed by hydrogen bonding of carboxylic
(
including 8-units of TBAm and 1–4-units of AAc) on average.
Molecular length of the 20-unit LP is shorter than melittin
26 amino acid peptide). Thus, the extremely high binding
22
acids and amides during the polymerization process and/or
hydrophobic interaction of tert-butyl groups and polymer main
chain might cause the steric hindrance for the melittin to access
the domain. Those intra-particle cross-links might be dramati-
cally limited in the case of LPs, since LPs were polymerized
without cross-linkers in the absence of strong intra- and inter-
polymer interaction by using good solvent (methanol instead of
(
capacity of LPs observed in this study may suggest that the LPs
recognized and captured limited sequence of melittin,
presumably c-terminal cation-rich fragment, as NPs of the same
18
composition did (Fig. 2b).
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.3. LP binding affinity of melittin
Journal of Materials Chemistry B
Table 2 Binding constant of the interaction between LP1000, LP300
,
2
and NPs
The apparent binding constant between LPs and melittin was
calculated by tting binding curves obtained by the 27 MHz
quartz crystal microbalance (QCM) to Langmuir isotherm as
Incorporated ratio
(mol%)
7,10
reported (Fig. S1†). A high binding constant (K
a
) greater than
ꢁ1
Polymer
TBAm
AAc
a
K (M )
6
ꢁ1
10 M
for LP300 consisting of relatively high ratio of both
8
7
7
TBAm and AAc was conrmed (Fig. 3a). However, little inter- LP1000-11
40
38
40
20
22
19
1.1 ꢀ 10
1.2 ꢀ 10
6.6 ꢀ 10
action was observed by QCM for all LPs containing only AAc or LP300-11
10
NP
TBAm.
The apparent binding constant increased dramatically by
increasing amount of AAc in LP300 containing 40% TBAm
Fig. 3c). LP300 containing 40% TBAm and 20% AAc had the
maybe because they are cross-linked and the exibility of
polymer chains are lower than those of LP1000
(
7
.
strongest binding with a binding constant of K
M
a
¼ 1.2 ꢀ 10
ꢁ1
. To explain the strong affinity, the average number of
monomer unit and AAc, that can be used to capture one
melittin, was calculated from the binding capacity of each LPs
3
. Conclusion
(
Fig. 3c). Fig. 3 indicates that LP300 containing 40% TBAm and In summary, we prepared a multi-functional linear polymer
0% AAc are capable of capturing one melittin with 4 carboxylic library having a two different molecular weights and range of
acids in addition to 8 tert-butyl group thru multi-point and functional groups. Only LPs containing both TBAm and AAc
2
multi-modal interaction, enabling the strong affinity.
were capable of neutralizing the toxicity of melittin. The
10
ꢁ1
The binding constant of LP300, LP1000, and NPs consisting maximum melittin-binding capacity was 1.5 g g , which was
of same ratio of functional monomers (40% TBAm and 20% approximately three times higher than that of the previously
AAc) were obtained and shown in Table 2. Interestingly, LP1000 reported NPs. In addition, amount of melittin with can be
8
ꢁ1
(
(
K ¼ 1.1 ꢀ 10 M ) showed even stronger affinity than NPs captured and neutralized by a gram of LPs was independent of
a
7
ꢁ1
6.6 ꢀ 10 M ), although, NPs showed stronger affinity than the molecular weight of LPs. Meanwhile, the binding constant
7
ꢁ1
LP (K ¼ 1.2 ꢀ 10 M ). It has been reported that exibility strongly depended on the M
w
of the polymers and the strongest
¼ 1.1 ꢀ 10 (M ) was found with LPs with a
3
00
a
8
ꢁ1
of polymer chain is important for the functionalized polymers affinity of K
a
to form stable polymer–protein complex through multipoint exible and relatively large molecular weight. Since, little has
14,15,23
interaction.
Since LP1000 has a larger molecular weight been reported about the inuence of the M and structure of
w
than LP300, it has a higher degree of freedom in its structure and synthetic polymers on their binding capacity and affinity with
more easily map onto melittin to form high affinity binding peptides, this study may be useful for the design of synthetic
sites than LP300. Although, NPs have a much higher molecular polymers ligands capable of strongly binding with biological
weight than LP1000, NPs showed weaker affinity than LP1000 molecules.
4. Experimental section
4
.1. Materials
AAc (Wako Pure Chemical Industries) was used aer purica-
tion by an alumina column (Al pH 6.8–7.8). NIPAm (Wako
2 3
O
Pure Chemical Industries) was used aer recrystallization from
benzene and n-hexane. TBAm (Wako Pure Chemical Industries)
was used as received. Preserved bovine blood (50% Alsever's
solution) was purchased from Nippon Bio-Test Laboratories
Inc. Melittin (from bee venom; molecular weight (M ): 2846 Da)
w
was purchased from SERVA. Biotinylated melittin (sequence;
Gly–Ile–Gly–Ala–Val–Leu–Lys–Val–Leu–Thr–Thr–Gly–Leu–Pro–
Ala–Leu–Ile–Ser–Trp–Ile–Lys–Arg–Lys–Arg–Gln–Gln–Lys(PEG4-
2 w
Biotin)–NH , M ; 3448.3 Da, purity; 98.2%) was purchased from
American Peptide Company. Other reagents were purchased
from Wako Pure Chemical Industries or Tokyo Chemical
Industry, Co., Ltd. and were used as received.
Fig. 3 (a) The melittin-binding constant of LP300 of various compo- 4.2. Synthesis of chain transfer agent
sitions. (b) Influence of the amount of AAc on the melittin-binding
capacity and binding constant of LP300 containing 40% TBAm. (c)
Average number of monomer units and average number of AAc that
are allowed for the LPs to capture one melittin.
The chain transfer agent, 3-benzylsulfanylthiocarbonylsulfanyl
22,24
propionic acid (BPATT), was synthesized as reported.
KOH
(12.9 g, 230 mmol) was dissolved in water (125 mL), and 3-
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ꢁ
1
mercaptopropanoic acid (10.0 mL, 115 mmol) was added and
A stock solution of the LPs (30 mg mL ) in DMSO was
the mixture was stirred for 4 h. Carbon disulde (24.3 mL, 253 prepared and diluted in PBS to the desired concentration. An
mmol) was added to the mixture, which was stirred at room 18 mM stock solution of melittin was prepared by dissolution in
temperature for 5 h. To the resulting solution, benzyl chloride milliQ water. Twenty microliters of melittin stock solution, 2 mL
ꢃ
(
27.5 mL, 115 mmol) was added and warm to 80 C. Aer stir- of an PBS solution of LPs, 30.4 mL of 5 times concentrated PBS,
ring for 12 h, the reaction mixture was diluted with chloroform and 101.6 mL of milliQ water were added into a 1.5 mL micro-
ca. 30 mL), followed by addition of 1 N HCl until the color of the tube and the mixture was pipetted ve times. Each sample was
(
solution changed from orange to yellow. The aqueous layer was prepared in triplicate. A solution of red blood cells (48 mL) was
extracted with chloroform (3 ꢀ 40 mL). The combined organic added to each sample and pipetted ve times. The mixture was
ꢃ
layer was washed with water (3 ꢀ 10 mL) and dried in vacuo. The incubated in a thermostat (MG-1200 EYELA) at 37 C for 30 min.
obtained yellowish solid was dissolved in ethyl acetate, followed Aer incubation, the samples were centrifuged at 800 G
by addition of hexane to give a yellowish solid. Aer ltration, (KUBOTA 1120) for 10 min. Immediately aer centrifugation,
1
BPATT was obtained in 69% yield. H NMR (JNM-ECP400) 10 mL of the supernatants were recovered then diluted 20-fold in
(
CDCl
3
): d 2.84 (t, 2H, CH
2
CO), 3.62 (t, 2H, CH
2
S), 4.61 (s, 2H, PBS in a 96-well plate. The absorbance (405 nm, Asample) of each
solution was measured by a plate reader (MULTISKAN JX:
Thermo electron). Samples using milliQ water instead of an
aqueous solution of melittin (A100%), and samples using DMSO
instead of the DMSO solution of LPs (A0%) were prepared as
CH –Ph), 7.27 (m, 5H, Ar–H).
2
4
.3. Synthesis of linear polymers
BPATT, NIPAm, TBAm, and AAc were dissolved in methanol and controls. The hemolytic activity inhibition ratio was calculated
mixed to obtain a total monomer concentration of 1.5 M and a as follows:
monomer : chain transfer agent ratio of 300 : 1 or 1000 : 1.
À Á
Asample ꢁ A
0%
Inhibition ratio % ¼
ꢀ 100
Next, 1.5 mL of the solution was added to a glass tube: outer
tube diameter was 10 mm: glass thickness was 2 mm. A cycle of
melting, vacuum, and freezing was repeated three times. The
tube was sealed using a gas burner and irradiated with 2.5 mW
A
100% ꢁ A0%
Inhibition curves were achieved by plotting inhibition ratio
against concentration of LPs. Binding capacity of LPs was
calculated from the initial slope of the inhibition curves.
ꢁ
2
cm ultraviolet light (High Power Xenon light source MAX-302:
ASAHI SPECTRA). The polymerization reaction was conducted
10
ꢃ
for 12 h at 70 C. Aer the reaction, the tube was opened and the
reaction was terminated by oxygen in the atmosphere. The
reaction solution was added dropwise into an excess amount
4
.5. Analysis of the binding constant by using a quartz
crystal microbalance (QCM)
(>30 mL) of diethyl ether or water. Polymers were recovered by
centrifugation (H-103n KOKUSAN). The conversion and the An Affinix Q4 QCM instrument (Initium Co. Ltd., Tokyo, Japan)
1
incorporation ratio of monomers were quantied using H- was used to quantify interactions between the NPs and melittin
7
,10
NMR (JNM-ECP400) (solvent: CD
ular weight: M , weight average molecular weight: M
polydispersity index (PDI ¼ M /M ) were conrmed by using gel for 10 min or longer. Surface of the gold electrode (substrate) on
3
OD). Number average molec- and control proteins.
A 1% sodium dodecyl sulfate (SDS)
n
w
, and solution (500 mL) was added to a QCM cell and allowed to stand
w
n
permeation chromatography (GPC): solvent; DMF/10 mM LiBr, the bottom of the cells, containing SDS solution, was wiped
ꢁ1

ow rate; 0.5 mL min , column; LF-804 Shodex, RI detector; RI- carefully by a cotton swab to remove dust. Aer washing the cell
031 plus (Jasco), UV detector; UV-970 (Jasco), pump; Pu-2080 with water, 2.5 mL of a fresh piranha solution (hydrogen per-
plus (Jasco), column oven; CO-965 (Jasco), auto sampler; AS- oxide : concentrated sulfuric acid (1 : 3)) was mounted onto the
057 (Jasco), degasser; DG-980-50 (Jasco), molecular weight gold substrate, allowed to stand for 10 min, and washed with
2
2
standard; polystyrene.
water. This piranha washing process was performed three times
to remove all organic matter from the gold surface. MilliQ water
(
(
200 mL) was added to the QCM cell soon aer it was washed
4
.4. Hemolysis inhibition experiments
note that the gold surface should not be dried aer the piranha
0
The red blood cells were puried from preserved bovine blood, washing). Subsequently, ethanol solution (0.1 M, 2 mL) of 3,3 -
in accordance with the following procedure. Preserved bovine dithiodipropionic acid (M : 210.26) were also added and the
6,9
w
blood (500 mL) was placed in a 1.5 mL microtube with 1 mL mixture was allowed to stand for 30 min or longer to carboxylate
phosphate buffered saline solution (PBS; 35 mM phosphate, pH the gold surface. The carboxylated QCM cell was washed with
7
.3, 150 mM NaCl). The suspension was mixed and subse- water. 50 mL of aqueous 1-ethyl-3-(3-dimethylaminopropyl)
quently centrifuged at 800 G (KUBOTA 1120) for 10 min. Aer carbodimide hydrochloride (EDC, M : 192.70) solution (100 mg
centrifugation, the supernatant was removed then 1 mL PBS mL ) and 50 mL of aqueous N-hydroxysuccinimide (NHS, M
w
ꢁ1
w
:
ꢁ
1
was added again. This procedure was repeated ve times. Aer 115.09) solution (100 mg mL ) was added to the QCM cells,
the nal centrifugation, 80 mL of the precipitated red blood cells and was allowed to stand for 30 min at room temperature. Aer
were recovered slowly by pipet and diluted four-fold in PBS. This washing by milliQ water, 50 mL of avidin (from hen egg white)
ꢁ1
suspension was used as the red blood cell solution and was solution (PBS, 1 mg mL ) was added and was allowed to stand
prepared prior to each experiment.
for 1 h at room temperature. Aer the avidin-immobilized
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Paper
Journal of Materials Chemistry B
substrate was washed with water, 500 mL of HEPES buffer
Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 8691; (b)
C. Cabaleiro-Lago, F. Quinlan-Pluck, I. Lynch, S. Lindman,
A. M. Minogue, E. Thulin, D. M. Walsh, K. A. Dawson and
S. Linse, J. Am. Chem. Soc., 2008, 130, 15437.
9 (a) Y. Yonamine, Y. Hoshino and K. J. Shea,
Biomacromolecules, 2012, 13, 2952; (b) Y. Yonamine,
K. Yoshimatsu, S. H. Lee, Y. Hoshino, Y. Okahata and
K. J. Shea, ACS Appl. Mater. Interfaces, 2013, 5, 374.
(
10 mM HEPES, pH 7.4) was added and the solution was set on
ꢃ
the Affinix Q4 instruments at 25 C. 1 mL of water solution of
biotinylated melittin (100 mM) was injected into the cell and the
time-course of frequency changes were measured. One hour
later, the melittin-immobilized QCM cell was washed with
water, replaced with 500 mL PBS, and set on the Affinix Q4
ꢃ
instruments at 37 C. Aer stabilizing the base line, PBS solu-
tion of LPs was injected consecutively into the cell and the time- 10 Y. Hoshino, H. Koide, K. Furuya, W. W. Haberaecker III,
course of frequency change were monitored. For each LPs
concentration, amount of LPs bound on the melittin-immobi-
S.-H. Lee, T. Kodama, H. Kanazawa, N. Oku and K. J. Shea,
Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 33.
lized sensor were plotted and the apparent binding constant K
11 S.-H. Lee, Y. Hoshino, A. Randall, Z. Zeng, P. Baldi, R. Doong
and K. J. Shea, J. Am. Chem. Soc., 2012, 134, 15765.
a
ꢁ
1
7,10
(M ) was calculated using the Langmuir isotherm.
12 Y. Hoshino, T. Kodama, Y. Okahata and K. J. Shea, J. Am.
Chem. Soc., 2008, 130, 15242.
Acknowledgements
1
3 Y. Hoshino, W. W. Haberaecker III, T. Kodama, Z. Zeng,
Y. Okahata and K. J. Shea, J. Am. Chem. Soc., 2010, 132,
13648.
We are grateful for nancial support from MEXT (25107726),
JSPS (23750193), JST (AS2321466 and AS231Z01490D), Otsuka
Chemical Co., Ltd. and Kao Foundation for Arts and Sciences. 14 Y. Hoshino, M. Nakamoto and Y. Miura, J. Am. Chem. Soc.,
012, 134, 15209.
5 M. Nakamoto, Y. Hoshino and Y. Miura, Biomacromolecules,
014, 15, 541.
2
1
1
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