Synthesis of poly(methyl methacrylate)-S/oc/c-poly(l-histidine) and its use as a hybrid silver nanoparticle conjugate

Article abstract of DOI:10.1166/jnn.2010.2954

Poly[(methyl methacrylate)-block-poly(L-histidine)] (PMMA-b-PHIS) was synthesized by combining atom transfer radical polymerization and living ring-opening polymerization of α-amino acid-N- carboxyanhydride. The resulting hybrid block copolymer forms reverse micelles in the mixture solution of water and N,N-dimethylformamide (DMF) and self-assembles into PHIS/PMMA core/shell spheres with controllable size in the range of 80 to 250 nm depending on the micellization temperature. The self-assembly of PMMA-b-PHIS was carried out in H₂0/DMF (3/7) mixture in the presence of AgN0₃. Reduction of the resulting Ag ions encapsulated inside of the reverse micelles yielded an attractive Ag nanoparticle core/polymer shell conjugate system. Copyright

Full text of DOI:10.1166/jnn.2010.2954

Copyright © 2010 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 10, 6948–6953, 2010
Synthesis of Poly(methyl methacrylate)-Block-
Poly(L-histidine) and Its Use as a Hybrid Silver
Nanoparticle Conjugate
∗
Nam Ho Shin, Jin Kyu Lee, Haiqing Li, Chang-Sik Ha, Yury A. Shchipunov, and Il Kim
The WCU Center for Synthetic Polymer Bioconjugate Hybrid Materials, Department of Polymer Science and Engineering,
Pusan National University, Busan 609-735, Korea
Poly[(methyl methacrylate)-block-poly(L-histidine)] (PMMA-b-PHIS) was synthesized by combining
atom transfer radical polymerization and living ring-opening polymerization of ꢀ-amino acid-N-
carboxyanhydride. The resulting hybrid block copolymer forms reverse micelles in the mixture solu-
tion of water and N,N-dimethylformamide (DMF) and self-assembles into PHIS/PMMA core/shell
spheres with controllable size in the range of 80 to 250 nm depending on the micellization tempera-
ture. The self-assembly of PMMA-b-PHIS was carried out in H O/DMF (3/7) mixture in the presence
2
of AgNO . Reduction of the resulting Ag ions encapsulated inside of the reverse micelles yielded
3
an attractive Ag nanoparticle core/polymer shell conjugate system.
Keywords: Amphiphilic Block Copolymer, Histidine-N-Carboxyanhydride, ATRP, Ring-Opening
Polymerization, Polypeptide, Self-Assembly.
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1
. INTRODUCTION
of molecular design. Monomer species are formed by
converting ꢀ-amino acids into NCAs, usually in a sin-
gle step, followed by solution polymerization where chain
length is controlled simply by monomer to initiator sto-
ichiometry. Subsequent batch wise addition of different
NCA monomers to the active chain-ends leads to multi-
block architectures with well-defined block lengths and
low polydispersity.
Amphiphilic block copolymers have received widespread
attention over the past few decades owing to the well-
known fact that they exhibit many different types of mor-
phologies in the bulk phase and in solution when placed
in a selective solvent.1 They give rise to diverse mor-
phologies due to their interesting supramolecular self-
–2
assembling behavior which imparts a number of unique
properties to them.3–5 Especially amphiphilic hybrid mate-
Recently developed controlled radical polymerization
(CRP) techniques such as atom transfer radical poly-
rials that contain both peptide and synthetic polymer
elements could synergistically combine the properties
merization (ATRP) and reversible addition–fragmentation
chain transfer (RAFT) polymerization can be used to
produce a number of end-functionalized polymers for
of the individual blocks and overcome several of their
limitations.6
6
–8
chemoselective reaction with nonnatural polypeptides.
Even though peptides are attractive since their synthesis
is very straightforward, the preparation of long sequences
that are essential to achieve hierarchical supramolecular
interactions is both tedious and very expensive. The prepa-
ration of well-defined materials from synthetic polypep-
tides is now feasible due to the invention of Co and Ni
initiators that allow the living ring-opening polymerization
The combination of CRP with living ROP of NCAs non-
natural protein engineering could result in unprecedented
control over polymer conjugation, resulting in precise syn-
thetic polymer bioconjugates for a variety of applica-
tions. The integration of bio-segments, which exhibit often
highly specific bio-functions, into the world of synthetic
polymers offers enormous possibilities for materials sci-
ence and biomedical science. Indeed, the resulting biocon-
jugates are tools for the direct translation of bio-inspired
science, leading already to interesting materials but also
contributing to the understanding of functions and sys-
temic behavior of biomacromolecules, biomaterials, and
(
ROP) of ꢀ-amino acid-N-carboxyanhydrides (NCAs).6
The versatility of this ROP, in particular the ability to
incorporate a large variety of amino acid monomers, both
natural and synthetic, results in unprecedented flexibility
∗
Author to whom correspondence should be addressed.
6
948
J. Nanosci. Nanotechnol. 2010, Vol. 10, No. 10
1533-4880/2010/10/6948/006
doi:10.1166/jnn.2010.2954

Shin et al.
Synthesis of PMMA-b-PHIS and Its Use as a Hybrid Silver Nanoparticle Conjugate
more complex bio-systems. In order to demonstrate a ver-
satility of synthetic polymer polypeptide hybrid materials,
we have combined ATRP and living ROP of NCA using
nickel initiator and utilized them for the stable encapsula-
tion of silver nanoparticle (AgNP).
2
. EXPERIMENTAL DETAILS
2
.1. Materials and Measurements
Methyl methacrylate (MMA) and solvents purchased from
Aldrich were purified according to the standard proce-
dures and stored under nitrogen. L-histine monomer with
blocking group, N -Boc-Nꢁimꢂ-benzyl-L-histidine (Boc-
ꢀ
His(Bzl)-OH), was obtained from Fluka. The cyclization
of Boc-His(Bzl)-OH was carried out to get N-benzyl-L-
9
histidine NCA · HCl. All the reactions were performed
under a purified nitrogen atmosphere using standard
1
13
Schlenk techniques. H NMR and C NMR spectra of
the compounds were recorded on a Varian Unity Plus-
3
00 spectrometer in CDCl or DMSO (d ꢂ as a solvent.
3 6
Scheme 1. Synthesis of poly(methyl methacrylate)-block-poly(L-
histidine) by combining atom-transfer radical polymerization with living
ring-opening polymerization of L-histidine-N-carboxyanhydride in the
presence of (depe)Ni(COD) complex.
Gel permeation chromatography (GPC) measurement was
carried out using a Waters 515 in DMF with a refractive
index detector calibrated by polystyrene standards. UV-vis
analysis was performed by Shimadzu UV-1650PC. Micel-
lization of block copolymers was performed using Bio-
Delivered by Publishing Technol oa tg yr o toom: Mt e cmM p ae sr at et urr eU nf oi vr e3rs di ta yy s. The mixture was precip-
rad thermal cycler. The size of polymeric micelles was
ꢀ
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itated in methanol, filtered and vacuum dried at 50 C.
measured by dynamic light scattering using a Malvern’s
Zetasizer Nano. Transmission electron microscope (TEM)
images were collected with a Philips EM400T TEM, at an
accelerating voltage of 80 kV.
Copyright: American Scientific Publishers
The resulting block copolymer (7) was deprotected by
1
3
hydrolysis to give PMMA-b-PHIS (8).
2.3. Fabrication of Polymeric Micelles and
2
.2. Synthesis of Poly(methyl
Ag-Polymer Conjugate
methacrylate)-Block-Poly(L-histidine)
The stable micelle solution was prepared by thermal cycler
(
PMMA-b-PHIS)
in DMF/H O mixture solution. In a typical process, 1 mg
2
The general procedures to prepare PMMA-b-
of poly(MMA77-b-HIS28ꢂ was dissolved in 0.7 mL of
DMF, and then 0.3 mL of deionized water was injected
into the copolymer solution. The resulting mixture was
PHIS were summarized in Scheme 1. The ATRP
ꢀ
of MMA was performed at 70
C
by using
ꢀ
CuBr/pentamethyldiethylenetriamine (PMDETA) system
heated to 110 C, and then slowly cooled down to a desired
1
0
ꢀ
and benzyl 2-bromopropanoate (1) as an initiator to
give PMMA (2). The bromide groups in the chain end
were converted to amine group by coupling reaction with
ethylenediamine. The resulting amine terminated PMMA
temperature (1 C/min) by using thermal cycler and kept
for 5 h at this temperature.
For fabrication of AgNP-polymer conjugates, 1 ꢄL
of AgNO3 solution (0.01 M) is mixed with 5 ꢄL of
poly(MMA -b-HIS ꢂ stock solution (20 mg/mL DMF),
(
3) was further reacted with alloc-L-leucine-N-hydroxy-
7
7
28
1
1ꢃ12
succinimidyl ester (4) to give 5.
The reaction of
35 ꢄL of DMF and 15 ꢄL of air-free water. The mixture
ꢀ
(
depe)Ni(COD) (COD = 1,5-cyclooctadiene) complex,
was then heated to 110 C for 2 h. It was then cool down
ꢀ
ꢀ
which has been in situ generated by reacting Ni(COD)
and 1,2-bis(diethylphosphino)ethane in an equivalent ratio
in THF (2 mL) for 15 min, with alloc-L-leucine ter-
minated PMMA (5) yielded macro-initiator (6) bearing
to 50 C at a rate of 1 C/min. To remove excess AgNO ,
3
the 5 batch of this solution was diluted with 2 mL of
water and centrifuged at 20,000 rpm for 30 min. This step
was repeated two more times and a concentrated polymer-
Ag ion conjugate solution was obtained. The same exper-
iments were repeated by changing the amount of AgNO3
solution to 5 ꢄL. The Ag ions encapsulated inside of the
micelles were further reduced to fine AgNPs by diffusing
1
2
amido-amidate nickelacycle end groups. In a typical
ROP of NCA, (0.1 g, 0.013 mmol of macro-initiator (6)
was added to N-benzyl-L-histidine NCA · HCl (0.34 g,
1
.25 mmol) dissolved in 5 mL of THF, and then stirred
J. Nanosci. Nanotechnol. 10, 6948–6953, 2010
6949

Synthesis of PMMA-b-PHIS and Its Use as a Hybrid Silver Nanoparticle Conjugate
Shin et al.
the reducing agent (NaBH ꢂ, resulting in the formation of
4
PMMA-b-PHIS encapsulating AgNP composite cores.
3
. RESULTS AND DISCUSSION
As a means of demonstrating a versatility of synthetic
polymer polypeptide hybrid materials, PMMA-b-PHIS
was synthesized by the combination of ATRP and living
ROP of NCA using nickel initiator (Scheme 1). Figure 1
1
shows the H-NMR spectrum of the resulting PMMA-b-
PHIS. The disappearance of benzylic blocking groups in
histidine moiety and appearance of characteristic peaks
in histidine and MMA blocks clearly show that well-
defined poly(MMA -b-HIS ꢂ is formed. The structure of
77
28
1
3
the block copolymer could also be confirmed by C-NMR
spectrum (not shown). In addition molecular weight (MW)
of PMMA increases from 8000 to 12000 after further
polymerization of his-NCA (Fig. 2). The resulting poly-
mer is characterized by low polydispersity index (1.25).
Two more PMMA-b-PHISs, poly(MMA -b-HIS ꢂ (M =
Fig. 2. GPC chromatograms of (a) PMMA (Mn = 8000) and
(
b) PMMA-b-PHIS [poly(MMA -b-HIS ꢂ] (M = 12000).
77
28
n
3
7
30
n
unspecific interactions. In the case of synthetic polymer–
polypeptide hybrids, however, this process is mediated by
the specific folding and organization properties of the pep-
tide sequence, which allows access to complex, hierar-
chically organized, and potentially functional materials
8
200; PDI = 1ꢅ27) and poly(MMA -b-HIS ꢂ (M =
2
5
35
n
7
600; PDI = 1ꢅ24), were also synthesized by similar proce-
dures using amido-amidate nickelacycle end groups func-
tionalized PMMA with different MW (see Table I for
detailed results).
that are difficult to generate from purely synthetic building
One reason that has driven part of the current inter-
15
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est in synthetic polymer–polypeptide hybrids is related
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We studied the self-assembly properties of
to the very specific self-assembly pr Co poe pr tyi er isg ht ht :a tA mc a en ri bc ea n Scientific Publishers
poly(MMA -b-HIS ꢂ block copolymer. This hybrid
7
7
28
programmed in the primary structure of the peptide seg-
ment. The self-assembly of common low MW amphiphiles
or high MW block copolymers is generally driven by
block copolymer can be considered a giant amphiphile in
which the PMMA chain represents the hydrophobic tail
and the PHIS chain acts as the hydrophilic head group.
The formation of micelles can be evidenced by means
of TEM analysis (Fig. 3). The contrast in Figure 3(a)
clearly indicates that stable core/shell spherical micellular
structure is formed. The average diameter of the resulting
micelles obtained at room temperature is around 250 nm.
It is interesting to note that the size of micelles is tunable
by changing the micellization temperature: i.e., the size
of micelles decreases with the increase of the temper-
ature, which can also be monitored by dynamics light
scattering (DLS) analysis (Figs. 3(c, d)). As a result the
ꢀ
average diameter decreases to 80 nm at 85 C (Fig. 3(b)).
The similar DLS analyses by changing the micellization
temperature carried out with poly(MMA -b-HIS ꢂ and
3
7
30
poly(MMA -b-HIS ꢂ yield similar results (Fig. 3(d)) and
2
5
35
no conspicuous change of the size of the micelle has been
observed according to the block size.
The core of the micelle consists of hydrophilic PHIS
blocks containing imidazole groups in them. The imida-
zole moieties, which are the driving factor of polymer self-
assembly, as well as, Ag-conjugation, make this a unique
system. The high affinity of the imidazole groups for the
Ag induces the self-organization of polymer molecules in a
reverse fashion (Scheme 2). By means of these functional
1
Fig. 1. H-NMR spectrum of poly(MMA -b-HIS ꢂ.
77
28
6
950
J. Nanosci. Nanotechnol. 10, 6948–6953, 2010

Shin et al.
Synthesis of PMMA-b-PHIS and Its Use as a Hybrid Silver Nanoparticle Conjugate
Table I. Polymerization results of ROP of N-benzyl-L-histidine NCA · HCl (BzHIS-NCA) initiated by amine terminated PMMAs of with different
ꢀ
molecular weights. Conditions: NCA/PMMA-NH /(depe)Ni(COD) = 100/1/1 (molar ratio), THF = 5 mL, temperature = 80 C, and time = 3 days.
2
Reactants
Poly(methyl methacrylate)-block-
poly(L-histidine)
No. of repeating unit
in block copolymer
a
PMMA
Amount
[g (mmol)]
BzHIS-NCA
(g)
BzHIS-NCA/
b
b
c
PMMA-NH
2
b
b
Sample code
Mn
PDI
MMA
HTD
Mn
PDI
Poly(MMA -b-HTD ꢂ
8000
4000
2800
1.20
1.19
1.20
0.1 (0.013)
0.1 (0.025)
0.1 (0.036)
0.34
0.68
0.98
100
100
100
77
37
25
28
30
35
12000
8200
7600
1.25
1.27
1.24
7
7
28
Poly(MMA -b-HTD ꢂ
3
7
30
Poly(MMA -b-HTD ꢂ
2
5
35
a
b
Amido-amidate nickelacycle end group functionalized PMMA to utilize as an initiator for ring-opening polymerization of BzHIS-NCA. Number average molecular weight
c
ꢁ
M ꢂ and polydispersity (PDI) of polymers determined by gel permeation chromatography (GPC). Molar feed ratio of N-benzyl-L-histidine NCA · HCl/amido-amidate
n
nickelacycle end group functionalized PMMA macroinitiator.
groups, the Ag ions are expected to be easily coordinated
and immobilized in the PHIS core matrix.
The formation of the polymer-Ag conjugate was moni-
tored by using TEM (Fig. 4). In order to check the effect
of Ag ion concentration on the formation of Ag-polymer
is intersting to note from the TEM observation that (1)
most of the spherical conjugates bear only one AgNPs in
them and (2) the size of AgNPs (∼38 nm) are indepen-
dent of the added amount of AgNO . These results demon-
3
strate that all nanoparticles formed inside of the micelle
are aggregated to form one bigger nanoparticle and that
conjugates, the amount of AgNO (0.01 M) was changed
3
from 1 to 5 ꢄL. At low concentration the relative percent-
age to form the Ag-polymer conjugates is low, so that there
are relatively large proportions of spherical micelles that
contain no AgNPs in them. The white (polymer) and black
(
AgNP) contrast was achieved by oxidizing the organic
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portion with aqueous (NH ꢂ MoO (2 wt%).
4
2
4
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The relative proportions of free micelles containing no
Copyright: American Scientific Publishers
AgNPs decrease with the increase of AgNO added. It
3
(a)
(b)
(c)
(d)
Fig. 3. Characterizations of PMMA-b-PHIS block copolymer micelle
solution. TEM image of stable spherical micellular structure obtained
ꢀ
ꢀ
at (a) 25 C and (b) 85 C; (c) representative particle size distribution
Scheme 2. The schematic representation of the self-assembly of
poly(methyl methacrylate)-block-poly(L-histidine) and the formation of
polymer-AgNP conjugate by the encapsulating of aggregated AgNPs
inside of the polymer micelle.
ꢀ
ꢀ
curves (by DLS) measured at 85 C and 25 C; and (d) average micelle
diameter versus temperature plots obtained by poly(MMA -b-HIS ꢂ,
poly(MMA -b-HIS ꢂ, and poly(MMA -b-HIS ꢂ.
37
30
2
5
35
77
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J. Nanosci. Nanotechnol. 10, 6948–6953, 2010
6951

Synthesis of PMMA-b-PHIS and Its Use as a Hybrid Silver Nanoparticle Conjugate
Shin et al.
Fig. 5. UV-vis absorbance of block copolymer-AgNP conjugate
obtained by adding different amounts of AgNO (0.01 M) in the mixture
3
consisting of 5 ꢄL of poly(MMA -b-HIS ꢂ stock solution (20 mg/mL
7
7
28
DMF), 35 ꢄL of DMF and 15 ꢄL of water: (a) AgNO3 (0.01 M) = 1,
b) 2, (c) 3, (d) 4, and (e) 5 ꢄL.
(
upon conjugation with this new class of PMMA-b-PHIS
hybrid polymeric system.
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Copyright: American S cA ie sne tri if ei cs Po fu bs yl i ns thh ee rt isc polymer block polypeptide (PMMA-
b-PHIS) copolymers were synthesized by the combination
Fig. 4. TEM images of block copolymer-AgNP conjugate obtained by
of atom transfer radical polymerization of MMA and living
ring-opening polymerization of his-NCA in the presence
of (depe)Ni(COD) complex. The resulting hybrid block
copolymers are self-assembled to form spherical micelles
with PHIS/PMMA core/shell structure due to their intrinsic
amphiphilicity in DMF/water mixture. The size of micelles
can be effectively controlled by varying micellization tem-
perature. By utilizing the chemical affinity of the PHIS
core to attract metal ions, we demonstrated PMMA-b-
PHIS polymers are a good hybrid material to construct
a facile and aggregation-free polymer AgNP conjugate.
Only a few polymeric systems have been developed for
the stabilization of nanoparticles and the successful appli-
cation of this new class of block copolymer as surface
stabilizers and a new preparative protocol should find wide
applications.
adding different amounts of AgNO in the mixture consisting of 5 ꢄL of
3
poly(MMA -b-HIS ꢂ stock solution (20 mg/mL DMF), 35 ꢄL of DMF
7
7
28
and 15 ꢄL of water: (a) AgNO (0.01 M) = 1, (b) 2, (c) 3, (d) 4, and (e)
3
5
ꢄL. The scale bar corresponds to 1 ꢄm.
the loaded amount of Ag ion in one micelle is similar
(Scheme 2).
The encapsulation using PMMA-b-PHIS is very useful
in that it does not require any initial surface modifica-
tion of AgNPs with common aliphatic ligands or thio-
lipids. The formation of polymer Ag conjugates could
also be evidenced by monitoring the UV-vis absorbance
of the resulting micelles which are corresponding to the
surface plasmon resonance energy of this polymer-AgNP
conjugate. As shown in Figure 5, the resulting conjugates
exhibit similar absorbance bands centred at 389 nm with
increasing intensities as the employed amount of AgNO3
Acknowledgment: This work was supported by the
Korea Research Foundation (KRF-D00422).
(0.01 M) is increased from 1 ꢄL to 5 ꢄL. All the samples
exhibit quite broad absorbance band, most probably due to
the corona effect of polymer shell. Moreover, the UV-vis
absorption of these nanoparticle conjugates after storage
over a prolonged time resembled to the newly synthesized
material. From all these experimental observations, it can
be concluded that the AgNPs are resistant to aggregation
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Received: 22 September 2008. Accepted: 13 August 2009.
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6953