The behavior of poly(amino acids) containing l -cysteine and their block copolymers with poly(ethylene glycol) on gold surfaces

Article abstract of DOI:10.1002/pola.26997

Poly(ethylene glycol) (PEG) is often used to biocompatibilize surfaces of implantable biomedical devices. Here, block copolymers consisting of PEG and l-cysteine-containing poly(amino acid)s (PAA's) were synthesized as polymeric multianchor systems for the covalent attachment to gold surfaces or surfaces decorated with gold nanoparticles. Amino-terminated PEG was used as macroinitiator in the ring-opening polymerization, (ROP), of respective amino acid N-carboxyanhydrides (NCA's) of l-cysteine (l-Cys), l-glutamate (l-Glu), and l-lysine (l-Lys). The resulting block copolymers formed either diblock copolymers, PEG-b-p(l-Glu_x-co-l-Cys_y) or triblock copolymers, PEG-b-p(l-Glu)_x-b-p(l-Cys)_y. The monomer feed ratio matches the actual copolymer composition, which, together with high yields and a low polydispersity, indicates that the NCA ROP follows a living mechanism. The l-Cys repeat units act as anchors to the gold surface or the gold nanoparticles and the l-Glu repeat units act as spacers for the reactive l-Cys units. Surface analysis by atomic force microscopy revealed that all block copolymers formed homogenous and pin-hole free surface coatings and the phase separation of mutually immiscible PEG and PAA blocks was observed. A different concept for the biocompatibilization of surfaces was followed when thiol-terminated p(l-Lys) homopolymer was first grafted to the surface and then covalently decorated with HOOC-CH₂-PEG-b-p(Bz-l-Glu) polymeric micelles. Copyright

Full text of DOI:10.1002/pola.26997

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The Behavior of Poly(amino acids) Containing L-Cysteine and Their Block
Copolymers with Poly(ethylene glycol) on Gold Surfaces
1
1
2
2
2
€
Rodolphe Obeid, Tracy Armstrong, Xiaoju Peng, Karsten Busse, Jorg Kressler,
Carmen Scholz1
1Department of Chemistry, University of Alabama in Huntsville, Huntsville, Alabama 35899
2Department of Chemistry, Martin Luther University of Halle-Wittenberg, D-06099 Halle (Saale), Germany
Correspondence to: C. Scholz; (E-mail: cscholz@chemistry.uah.edu)
Received 12 September 2013; accepted 21 October 2013; published online 14 November 2013
DOI: 10.1002/pola.26997
ABSTRACT: Poly(ethylene glycol) (PEG) is often used to biocom-
patibilize surfaces of implantable biomedical devices. Here,
block copolymers consisting of PEG and L-cysteine-containing
poly(amino acid)s (PAA’s) were synthesized as polymeric mul-
tianchor systems for the covalent attachment to gold surfaces
or surfaces decorated with gold nanoparticles. Amino-
terminated PEG was used as macroinitiator in the ring-opening
polymerization, (ROP), of respective amino acid N-carboxyan-
hydrides (NCA’s) of L-cysteine (L-Cys), L-glutamate (L-Glu), and
L-lysine (L-Lys). The resulting block copolymers formed either
diblock copolymers, PEG-b-p(L-Glux-co-L-Cysy) or triblock
copolymers, PEG-b-p(L-Glu)x-b-p(L-Cys)y. The monomer feed
ratio matches the actual copolymer composition, which,
together with high yields and a low polydispersity, indicates
that the NCA ROP follows a living mechanism. The L-Cys
repeat units act as anchors to the gold surface or the gold
nanoparticles and the L-Glu repeat units act as spacers for the
reactive L-Cys units. Surface analysis by atomic force micros-
copy revealed that all block copolymers formed homogenous
and pin-hole free surface coatings and the phase separation of
mutually immiscible PEG and PAA blocks was observed. A dif-
ferent concept for the biocompatibilization of surfaces was fol-
lowed when thiol-terminated p(L-Lys) homopolymer was first
grafted to the surface and then covalently decorated with
C
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HOOC-CH2-PEG-b-p(Bz-L-Glu) polymeric micelles.
2013 Wiley
Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52,
248–257
KEYWORDS: block copolymers; poly(amino acids); poly(ethylene
glycol); solid supports
PEG to proteins, drugs, polynucleotides, and other biopoly-
mers, where it provides a stealth character to the resulting
conjugates and their aggregates.11–15 Equally, surfaces grafted
with PEG will also exhibit biocompatibility.16 When exposed to
an aqueous environment, PEG coordinates with water mole-
cules, thereby preventing direct interactions between proteins
and the PEGylated surface or other PEGylated conjugates.17,18
INTRODUCTION The functionality and stability of biomedical
devices depends on their interactions with the surrounding
bioenvironment.1–4 Therefore, the molecular architecture of
the interface needs to be designed in such a way that it will
promote biocompatibility. Gold layers or gold nanoparticles
deposited onto the surface of biomedical devices may be
exploited as an adhesion layer to which biocompatible poly-
mers can be attached covalently by using the reactivity of
thiol groups toward gold. The naturally occurring L-cysteine
(L-Cys) carries a pendant thiol group, which readily forms
covalent linkages to gold surfaces.5 L-Cys can be homopoly-
merized to poly(L-cysteine), p(L-Cys) or copolymerized with
other amino acids to form either block or random copoly-
mers, thus conveying a reactivity toward gold to the result-
ing poly(amino acid)s (PAA’s).6–8
When block copolymers are formed between PEG and p(L-
Cys)-containing PAA’s, the biocompatibility of the PEG is
combined with the reactivity of p(L-Cys). Furthermore,
copolymerizing L-Cys with other amino acid repeat units, as
opposed to end-capping the polymer-chains with a single
thiol group, generates multiple anchor sites along the copoly-
mer chain that become available for the covalent attachment
of the block copolymer to a surface. When used in biomedi-
cal applications, PAA’s are inherently biocompatible and bio-
degradable as they consist of naturally occurring amino
acids, and PEG with a sufficiently low-molecular weight will
cleared via renal excretion.19–24
In general, biocompatibility is achieved, when surfaces are ren-
dered hydrophilic. Poly(ethylene glycol) (PEG) proved to be a
superior biomaterial that is capable of conveying biocompati-
bility.9,10 PEGylation is defined as the covalent attachment of
Additional Supporting Information may be found in the online version of this article.
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2013 Wiley Periodicals, Inc.
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This project was originally motivated by research into retinal
prostheses which are implanted into the subretinal space and
need to maintain their electrical activity while being biocom-
patible.25 Implanting electronic devices into living organisms
poses two challenges: (i) biocompatibility of the implant with
the surrounding tissue and (ii) the edema caused by the sur-
gical trauma. This study focuses on the synthesis and deposi-
tion behavior of block copolymers capable of conveying
biocompatibility and also offers a path to decorating biocom-
patible polymer brushes with potential drug delivery systems
that could carry drugs to alleviate the consequences of surgi-
cal trauma. It is inherently difficult to form dense polymer
brushes on surfaces using a “grafting to” method. Only lim-
ited graft densities can be achieved by “grafting to” methods
because of the concentration gradient that builds up by the
already attached chains, which provides a strong kinetic
impediment against additional grafting.26,27 In addition, the
large excluded volume that PEG exerts in an aqueous envi-
ronment further hinders the formation of dense PEG brushes
in “grafting to” procedures. Therefore, a multipoint attach-
ment using an anchoring polymer that in itself is biocompati-
ble was chosen here. Specifically, amino-terminated PEG was
used as the macroinitiator for the ring-opening polymeriza-
tion (ROP) of amino acid N-carboxyanhydrides (NCA’s). L-Cys-
containing PEGylated PAA’s were synthesized, where the PAA
block constitutes either a random copolymer of L-Cys and L-
glutamate (L-Glu) or a block copolymer of the two. These
block copolymers were coated onto gold surfaces or surfaces
with gold nanoparticles. Furthermore, p(L-lysine), (p-(L-Lys))
terminated with a thiol group on one chain end was depos-
ited onto gold surfaces and covalently decorated with poly-
meric micelles, which were also produced from PEGylated
PAA’s. The morphology of the resulting surfaces was investi-
gated by atomic force microscopy (AFM).
aqueous methanol solution 9:1 (v/v) of H2O: MeOH. Water
was deionized with a Millipore Milli-Q system.
Instrumentation
All Proton Nuclear Magnetic Resonance, 1H NMR, spectra were
recorded on a Varian Unity Inova 500 (500 MHz) spectrometer
equipped with a 5-mm triple resonance inverse detectable probe.
DMSO-d6, CDCl3, and D2O were used as solvents. Gel permeation
chromatography (GPC) was performed on a Waters Breeze GPC
system consisting of a 1515 isocratic pump, a TSK-gel a-M (parti-
cle size 13 mm, exclusion limit 1 3 107 g mol21 for polystyrene
in DMF), and a TSK-gel a-3000 (particle size 7 mm, exclusion
limit 1 3 105 g mol21 for polystyrene in DMF) (Tosoh Bio-
science) column, and a 2414 refractive index detector under the
following conditions: injection volume, 100 mL; flow rate, 0.5 mL
min21; eluent, DMF; temperature, 50 ꢀC, PS (purchased from
Shodex) as the calibration standards. Dynamic light scattering
(DLS) measurements on the polymeric aggregates using a 90Plus
particle size analyzer (Brookhaven Instruments) were described
previously.28 Briefly, solutions for DLS measurements ranging in
concentration from 0.25 to 5.0 g L21 were obtained from a
HOOC-CH2-PEG-b-p(Bz-L-Glu)n stock solution. Five measurements
of one min each were carried out and averaged.
X-ray scattering measurements were performed on an EMPY-
REAN diffractometer from PANanalytica, B.V., Almelo, The
Netherlands in point focus geometry using a combination of
crossed slits (0.2 3 1.0 mm2) on the incident side and a pro-
grammable anti-scatter slit with a fixed divergence of 0.5ꢀ in
front of a position sensitive PIXEL detector. The wavelength
used was CuKa radiation (k 5 0.154 nm), monochromatized
with Nickel filter. The measurements were carried out at
room temperature over the range 2h 5 15ꢀ to 30ꢀ in incre-
ments of 0.026ꢀ.
Surface morphologies were studied using an atomic force
microscope NanoWizard (JPK Instruments, Germany) work-
ing in tapping mode with silicon cantilevers. Cantilevers
were of type Arrow (NanoWorld, Switzerland) and had a res-
onance frequency of about 285 kHz and a force constant of
EXPERIMENTAL
Materials
All chemicals were purchased from Sigma-Aldrich Chemicals
St. Louis, MO and used as received, unless otherwise stated.
Triphosgene was purchased from Tokyo Chemical Industry,
Japan. (1S)-(-)-alpha-Pinene was purchased from Fisher Scien-
tific, Waltham, MA. Alpha-carboxymethyl-x-amino PEG, COOH-
CH2-PEG114-NH2 (Mw 5 5000 g mol21) and a-methoxy-x-
amino PEG, CH3-O-PEG114-NH2 (Mw 5 5000 g mol21) were
purchased from LaysanBio, Huntsville, AL. Gamma-benzyl-L-
glutamate (Bz-L-Glu) was purchased from Bachem, Torrence,
CA, and stored at room temperature and used as received, S-
benzyloxycarbonyl-L-cysteine (Bz-O-L-Cys) was purchased from
Sigma Aldrich Chemicals, St. Louis, MO and from Chengdu Uni-
biochem, China, and stored at 220 ꢀC in a sealed container
and e-trifluoroacetyl-L-lysine (TFA-L-Lys) was purchased from
Chem-Imex International, Wood Dale, IL and stored at room
temperature. Acetonitrile was dried by reflux over CaH2 under
a dry nitrogen atmosphere and subsequently distilled prior to
use. Dimethylformamide (DMF) was distilled under vacuum
and over CaH2. Methanolic potassium carbonate was prepared
by the addition of 250 mg potassium carbonate to 40 mL of an
about 42 N m21
.
Gold-Coated Silicon Wafers
Silicon wafers were purchased from Silicon Valley Microelec-
tronics, Inc. Gold coated wafers were obtained from Cornell Uni-
versity through the Boston Retinal Implant Project. They were
prepared by depositing a 50 nm layer of titanium, followed by
the deposition of a 50 nm layer of gold by electron beam evapo-
ration in a Chemical Vapor Coating 6-pocket e-beam evaporator.
Silicon wafers with Au nanoparticles with a diameter of 200–
400 nm were kindly provided by Dr. Chengdu Liang of Oak
Ridge National Laboratory. A previously described solution-
based technique for the synthesis of gold nanoparticles was
modified to allow for their synthesis on solid substrates.29,30
Synthesis of Protected NCAs
NCA of protected amino acids, were synthesized in dry ethyl
acetate (EtOAc) in the presence of triphosgene. Respective
amounts (0.05 or 0.1 g mL21) of c-benzyl-L-glutamic acid (Bz-L-
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TABLE 1 Summary of PEGylated PAA’s and Their Properties
Actual PAA
Composition
by 1H NMR
Yield
(%)
Mn by NMRb
mol21
g
PDI by
GPC
#
Polymera
1
2
3
4
5
6
7
8
9
PEG-b-p(Bz-L-Glu)50-b-p(Bz-O-L-Cys)10
PEG-b-p(Bz-L-Glu)100-b-p(Bz-O-L-Cys)10
PEG-b-p(Bz-L-Glu)100-b-p(Bz-O-L-Cys)20
57/9
101/7
108/14
50/8
102/7
100/19
29
87
90
88
91
86
89
87
78
75
19,700
29,100
32,300
18,000
29,400
31,600
9500c
7700
1.16
1.17
1.14
1.26
1.34
1.33
1.27
1.24
1.26
PEG-b-p(Bz-L-Glu50-co-Bz-O-L-Cys10
)
PEG-b-p(Bz-L-Glu100-co-Bz-O-L-Cys10
PEG-b-p(Bz-L-Glu100-co-L-Bz-O-Cys20
p(TFA-L-Lys)30-SH
)
)
HOOC-PEG-b-p(Bz-L-Glu)12
HOOC-PEG-b-p(Bz-O-L-Cys)16
10
13
8500
a
b
PEG: a-methoxy-x-amino PEG and a-carboxlymethyl-x-amino PEG,
Mn of block copolymers with the protected amino acid repeat units
respectively, with a molecular weight of 5000 g mol21 was used for all
experiments. PDI: polydispersity index.
(p(Bz-L-Glu) and p(Bz-O-L-Cys)) as determined by 1H-NMR
spectroscopy.
c
Synthesized as dimer from cysteamine.
Glu), e-TFA-L-lysine (TFA-L-Lys), or S-benzyloxycarbonyl-L-cyste-
ine (Bz-O-L-Cys) were suspended in freshly distilled EtOAc, 2.1
equiv. of a-pinene and 0.5 equiv. of triphosgene were added.
The reaction mixture was heated at reflux (105–110 ꢀC) until it
became transparent, which took, depending on the a-amino
acid, between 2.5 and 4.5 h. The mixture was cooled to room
temperature, filtered, and the filtrate was concentrated under
reduced pressure, and then crystallized in EtOAc/hexane. The
obtained NCAs were recrystallized from a mixture of EtOAc/
CO2 evolved, at which point 4.1 3 1024 mol of Bz-O-L-Cys-NCA
was added. The polymerization was conducted similar to the
method described by Wooley;31 however, here the polymeriza-
tion was performed under a blanket of Argon. Periodically, the
pressure that built due to the CO2 formation was checked via
the bubbler and released via the Schlenk line. The polymeriza-
tion continued for another 48 h, and the end point was again
determined when CO2 evolution had ceased. The PEG-b-p(Bz-L-
Glu)100-b-p(Bz-O-L-Cys)10 triblock copolymer was isolated by
precipitation into diethyl ether and purified by dialysis against
distilled water for 3 d using a Spectra/Por cellulose ester (CE)
ꢀ
hexane cooled to 0 C and then vacuum-dried. Yield: 86–93%.
1H NMR for Bz-O-L-Cys: (DMSO-d6, 500 MHz at 35 ꢀC): d/
ppm 5 3.34 (2H, CH2-S-CO), 4.74, (1H, CO-CH(CH2)-NH), 5.29
(2H, -O-CH2-Benzyl), 7.36 (5H, aromatic H), 9.129.24 (1H, -CO-
CH-NH-). 1H-NMR for TFA-L-Lys: (DMSO-d6, 500 MHz at 35 ꢀC):
d/ ppm 5 1.36 (2H, CH-CH2-CH2), 1.49 (2H, CH-CH2-CH2-CH2-),
1.73 (2H, CH-CH2-CH2-CH2-CH2-), 3.17 (2H, CH2-NH-CO-CF3),
4.43, (1H, CO-CH(CH2)-NH), 9.08 (1H, NH-CO-CF3), 9.40 (1H,
membrane (Molecular weight Cut-off, (MWCO): 3500 g mol21
)
and isolated by freeze-drying. Yield: 90% 1H NMR (DMSO-d6,
500 MHz):d/ppm 5 3.6–3.7(PEG, 4H, -O-CH2CH2-), 2.81 (3H,
CH3-O-), 1.89 (2H, CH-CH2-CH2-COO-), 2.21 (2H, CH-CH2-CH2-
COO-), 5.15 (2H, -O-CH2-C6H5), 7.18 (5H, CH2-C6H5-), 4.97 (2H,
S-COO-CH2- C6H5-), 3.83–4.19 (1H, -CO-CH(CH2)-NH-), 7.95
(-CO-CH-NH-) see: Supporting Information.
1
CH-NH-CO). Lastly, H-NMR for Bz-L-Glu: (DMSO-d6, 500 MHz
ꢀ
at 35 C): d/ ppm 5 1.93 (2H, CH-CH2-CH2-CO), 2.04 (2H, (CH-
Diblock copolymers in which the PAA block constitutes a ran-
dom copolymer, PEG-b-p(Bz-L-Glux-co-Bz-O-L-Cysy), were syn-
thesized as described above, except that the Bz-L-Glu-NCA and
Bz-O-L-Cys-NCA monomers were mixed prior to the addition
of the CH3-O-PEG-NH2 macroinitiator. The polymerization was
conducted for 4 d, and the yield was between 86 and 91%.
CH2-CH2-CO), 4.45 (1H, CO-CH(CH2)-NH), 5.09 (2H, -O-CH2-
C6H5), 7.36 (5H, CH2-C6H5), 9.08 (1H, -CO-CH-NH-) (Supporting
Information).
Synthesis of Methoxy-PEG-b-PAA Block Copolymers: (i)
Methoxy-PEG-b-Poly(L-Glutamate-b-L-Cysteine),
PEG-b-p(L-Glu)x-b-p(L-Cys)y and (ii) Methoxy-PEG)-
b-Poly(L-Glutamate-co-L-Cysteine), PEG-b-p(Glux-co-Cysy)
PEGylated PAA triblock copolymers were obtained by ROP of
the respective protected NCA’s using CH3-O-PEG-NH2 with a
molecular weight of 5000 g mol21 as macroinitiator. The syn-
thesis is exemplified for PEG-b-p(L-Glu)100-b-p(L-Cys)10: CH3-O-
PEG-NH2 (3.5 3 1025 mol) was dissolved in 5 mL of 0.2 M
urea/DMF and added with a syringe to a solution of Bz-L-Glu-
NCA (4.1 3 1023 mol) in 0.2 M urea/DMF (5 mL) kept under a
flow of dry argon. The polymerization was conducted at room
temperature for 72 h. The formation of the diblock (PEG-b-
p(Bz-L-Glu)100) was determined to be complete when no more
Synthesis of Cysteamine–Poly(e-TFA-L-Lysine)
(p(TFA-L-Lys)-SH)
The p(TFA-L-Lys)-SH homopolymer was obtained by ROP of
TFA-L-Lys-NCA using cysteamine [S-(CH2)2-NH2)]2 as initiator.
Cysteamine (6.5 3 1025mol) was dissolved in 5 mL of 0.2 M
urea/DMF and added with a syringe to the TFA-L-Lys-NCA
solution (2.5 3 1023 mol) in 0.2 M urea/DMF (25 mL) kept
under dry argon. The polymerization was conducted for 3 d
at room temperature. The p(TFA-L-Lys)-SH polymer was iso-
lated by precipitation into n-hexane. Next, the polymer was
dissolved in N-methyl-2-pyrrolidone and dialyzed against
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with a molecular weight of 5000 g mol21 as macroinitiator.
HOOC-CH2-PEG-NH2 (3.5 3 1025 mol) was dissolved in 5 mL
of 0.2 M urea/DMF and added with a syringe to a solution of
(Bz-L-Glu-NCA (5.5 3 1024 mol) in 0.2 M urea/DMF (5 mL)
kept under dry argon. The polymerization was conducted at
room temperature for 48 h. The HOOC-PEG-b-p(Bz-L-Glu)n
copolymer was isolated by precipitation into diethyl ether and
purified by dialysis against distilled water for 3 d using a
Spectra/Por CE membrane (MWCO: 3500 g mol21) and
isolated by freeze-drying. 1H NMR (DMSO-d6, 500 MHz):
d/ppm5 1.8 (2H, CH-CH2-), 2.35 (2H, CH-CH2-CH2-), 3.5 (PEG,
4H, -O-CH2CH2-), 3.9(1H, -CO-CH(CH2)-NH-, 5.0 (2H, -O-CH2-
C6H5), 7.27(5H, CH2-C6H5-), 8.1 (1H, -CO-CH-NH-).
SCHEME 1 Synthesis of PEGylated PAA’s using a-methoxy-x-
amino PEG as macroinitiator. Diblock copolymers with the fol-
lowing structure: PEG-b-p(L-Glux-co-L-Cysy) are obtained when a
mixture of the amino acid NCA’s is polymerized, triblock copoly-
mers with the following structure: PEG-b-p(L-Glu)x-b-p(L-Cys)y are
obtained when the amino acid NCA’s are added successively.
Self-Assembly of HOOC-PEGn-b-p(Bz-L-Glu)12
Polymeric aggregates were formed when the block copoly-
mer was dissolved directly in water as previously
described.24 Briefly, stock solutions (5.0 g L21) were pre-
pared by stirring the copolymer for at least 12 h in distilled
water. This stock solution was diluted to concentrations
ranging between 0.25 and 2.5 g L21, the resulting solutions
were filtered through a 0.45-mm Millex Millipore polyvinyli-
dene fluoride (PVDF, Durapore) filter.
water for 3 d using a Spectra/Por CE membrane (MWCO:
1
2000 g mol21) and isolated by freeze-drying. Yield: 87%. H-
NMR (DMSO-d6, 500 MHz):d/ppm 5 3.40–3.57 (4H, S-CH2-
CH2-N(H), 2.91 (2H, CH2-CH2-N(H), 1.42–1.73 (6H, CH-CH2-
CH2CH2-CH2), 4.26 (1H, -CO-CH(CH2)-NH-).
Deprotection of Cysteamine–Poly(e-TFA-L-Lysine)
(p(TFA-L-Lys)-SH) and Methoxy-PEG-b-PAA Block
Copolymers
Synthesis of Carboxymethyl-PEG-b-(c-Benzyl-L-Glutamate),
HOOC-CH2-PEG-b-p(Bz-L-Glu)n
The HOOC-CH2-PEGn-b-p(Bz-L-Glu)n block copolymer was
obtained by ROP of Bz-L-Glu-NCA using HOOC-CH2-PEG-NH2
Between 100 and 500 mg of the polymer or copolymer,
respectively, were suspended in a mixture of 9:1 (v/v) of
FIGURE 1 1H NMR spectrum of deprotected PEG-b-p(L-Glu100-co-L-Cys10) in the front, and protected PEG-b-p(Bz-L-Glu100-co-Bz-O-L-
Cys10) in the back. Chemical shift differences are due to the use of D2O used for the deprotected sample versus DMSO-d6 for the
protected sample. The inset shows the complete removal of the benzyl and the benzyloxylcarbonyl groups from the cysteine and
glutamate repeat units. Additionally, all methylene protons associated with the protective groups are also no longer present in the
spectrum in the front.
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H2O and MeOH with 250 mg K2CO3. The suspension was
heated to reflux. During the course of the reaction, the initial
suspension turned into a homogeneous solution, as depro-
tected PAA’s are sufficiently hydrophilic to render the poly-
mer or copolymer water-soluble. After 4–8 h, depending on
the PAA chain length, the reaction mixture was cooled to
room temperature and filtered. The filtrate was purified by
dialysis against distilled water for 2 d using a Spectra/
Por membrane (MWCO: 2000 g mol21), the resulting poly-
mer was isolated by freeze-drying. In the course of this
hydrolysis, protected p(TFA-L-Lys)-SH was converted into its
deprotected derivative p(L-Lys)-SH, similarly, p(Bz-L-Glu)
and p(Bz-O-L-Cys) were converted into p(L-Glu) and
p(L-Cys).Yield: 90–95%.
Surface Coating
The gold surfaces were cleaned prior to use by dipping
them in a piranha solution consisting of 70% concentrated
sulfuric acid and 30% hydrogen peroxide, followed by copi-
ous rinsing with deionized water, and drying in a flow of
nitrogen.
Immobilization of PEG-b-p(L-Glu-b/co-L-Cys) block
copolymers onto Au-substrates and Au-nanoparticles
present on silicon wafers
Deprotected PEGylated PAA’s were dissolved in 1 mL of
deionized H2O to yield a 50 mM concentration and then
deposited onto 1.0 3 1.0 cm squares cut from a gold-coated
silicon wafer. The substrate was placed into a vial, and gently
shaken at 150 rpm for approximately 12 h. The substrates
were removed from the solution and first rinsed with copi-
ous amounts of distilled water, followed by a rinse with 70%
ethanol. Coated substrates were dried in a stream of argon
gas.
Preparation of an self-assembled p(L-Lys)-SH monolayer
on gold substrate
The aqueous p(L-Lys)-SH solution (0.1 wt%) was prepared
by direct dissolution of the polymer in distilled water and fil-
tered through 0.45-mm Millipore PVDF filters prior to use.
The gold-substrate, 1.0 3 1.0 cm squares cut from the Au-
coated Silicon wafer, was incubated in the aqueous p(L-Lys)-
SH solution for at least 12 h. The substrate was then
removed, rinsed with water and 70% ethanol, and dried
under a steam of argon.
Covalent attachment of HOOC-PEG-b-p(Bz-L-Glu)n
aggregates to p(L-Lys), immobilized on Au-substrates
An aqueous solution of the HOOC-CH2-PEG-b-p(Bz-L-Glu)n
aggregates32 in EDC/NHS (N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide/N-hydroxysuccinimide) (0.5%/1%/0.5%
wt%, respectively) was freshly prepared at pH 6.5 and added
to the p(L-Lys)-coated substrate to initiate the coupling reac-
tion between the carboxyl groups that formed on the corona
of the aggregates and the amino groups of the p(L-Lys) that
extend from the substrate surface. The vials were gently
shaken and after 24 h, the substrate was transferred to a
clean empty vial rinsed with water to remove unreacted
polymeric aggregates and dried under a steam of argon.33
SCHEME 2 (a) Shows the multipoint attachment of a PEGy-
lated PAA chain to a gold surface. The PAA block consists
either of a block copolymer of p(L-Glu)x-b-p(L-Cys)y depicted
here or
a copolymer of p(L-Glux-co-L-Cysy). (b) Shows the
attachment of p(L-Lys)n oligomers to a gold surface via their
terminal thiol group, followed by the covalent attachment of
reactive polymeric micelles or aggregates, here: HOOC-CH2-
PEG-b-p(Bz-L-Glu)n. (c) Shows the attachment of a PEGylated
PAA chain to gold nanoparticles. The PAA block here consists
of a random copolymer of p(L-Glux-co-L-Cysy).
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FIGURE 2 5 3 5-mm AFM images of a gold surface coated with PEG-b-p(L-Glu)100-b-p(L-Cys)20. (a) Height image and (b) Phase
image with zoomed area.
FIGURE 3 5 3 5-mm AFM image (left: phase image, right: height image) of a gold surface coated with PEG-b-p((L-Glu)50-co-
(L-Cys)10) (a and b) and of PEG-b-p((L-Glu)100-co-(L-Cys)20) (c and d).
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FIGURE 4 (a) 10 3 10-mm AFM phase image of gold nanoparticles with diameter ranging between 200 and 400 nm coated with
PEG-b-p((L-Glu)50-co-(L-Cys)10). (b) 5 3 5-mm AFM phase image of PEG-b-p((L-Glu)50-co-(L-Cys)10). (c) For comparison, uncoated
gold nanoparticles are shown in the 10 3 10-mm AFM phase image.
Macroinitiators are facile for NCA ROP’s37 and PEGylated
RESULTS AND DISCUSSION
PAA block copolymers were synthesized by ROP of respec-
Results of Polymer Synthesis
tive amino acid-NCA’s using CH3-O-PEG-NH2 with a molecu-
NCA’s of amino acids were produced following established
lar weight of 5000 g mol21 as macroinitiator. Diblock
copolymers were synthesized when the amino acid NCA’s
were added as a mixture to the macroinitiator, yielding block
copolymers with the PAA block consisting of a random PAA
procedures based on the Fuchs-Farthing method.34,35 All
ROP’s of amino acid NCA’s reported here were conducted
under anhydrous conditions at room temperature in the
presence of urea. Previous research showed that urea effi-
ciently suppresses the formation of secondary structures
(b-sheets) between the nascent chains by breaking hydrogen
bonds between polymer chains in the early stages of the
polymerization.36 Reaction times varied between 48 and 120
h, depending on PAA-block length and increased with
increasing PAA block length. CO2 was released periodically
from the reaction mixture, and the polymerization was con-
sidered to be complete, when no more CO2 developed within
the reaction vessel.
copolymer:
PEG-b-p(Bz-L-Glux-co-Bz-O-L-Cysy).
Triblock
copolymers were synthesized when the amino acid NCA’s
were added successively to the polymerization, with Bz-L-
Glu-NCA added first, followed by Bz-O-L-Cys-NCA after the
formation of PEG-b-p(Bz-L-Glu) was completed, thus yielding:
PEG-b-p(Bz-L-Glux)-b-p(Bz-O-L-Cysy). Completion of the first
polymerization was determined by the lack of CO2 develop-
ment. Adding a second monomer, here Bz-O-L-Cys-NCA, to
the polymerization mixture restarts the polymerization,
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indicating that the chain end remained living. The analysis of
the block copolymers (Table 1, experiments 1–6) shows that
the actual polymer composition as determined by 1H NMR
spectroscopy matches the monomer feed ratio of the amino
acid-NCA’s used for polymerization. Copolymers were
obtained in yields greater than 85%, their polydispersity
ranged between 1.1 and 1.3, as determined by GPC. The
reported molecular weights were determined by 1H NMR
spectroscopy and reflect the molecular weights of the
copolymers with protected amino acid repeat units. The very
good match between the monomer feed ratio and actual
copolymer composition together with sufficiently high yields
and a low polydispersity indicate that the NCA ROP follows
a living mechanism, see Scheme 1, which is also in agree-
ment with the findings of Gibson and Cameron.38
FIGURE 5 WAXS data of PEG-b-p((L-Glu)50-co-(L-Cys)10) (top),
Homopolymers of TFA-L-Lys-NCA were synthesized using
cysteamine as initiator to yield p(L-Lys)-S-S-p(L-Lys), Table 1,
Experiment 7. The resulting homopolymer has 30 p(L-Lys)
repeat units, when exposed to a gold surface, the disulfide
bonds of cysteamine break, thereby producing p(L-Lys)-SH
grafted chains with approximately 15 repeat units.
PEG-b-p(L-Glu)100-b-p(L-Cys)10 (middle), and PEG-b-p((L-Glu)100
-
co-(L-Cys)20 (bottom). The curves are shifted vertically for
)
more clarity. When the PAA block has only a combined 60
repeat units, PEG is able to crystallize as indicated by the two
Bragg reflections at 19.2 and 23.4ꢀ, but with higher amount of
PAA, only amorphous halos are observed.
Homopolymers of p(TFA-L-Lys)-SH and PEGylated di- and tri-
block copolymers (PEG-b-p(Bz-L-Glux-co-Bz-O-L-Cysy) and PEG-
b-p(Bz-L-Glux)-b-p(Bz-O-L-Cysy)) were deprotected so that their
functional groups can undergo further chemical reactions. The
progress of the deprotection reactions was followed spectro-
scopically, and deprotection reactions were considered com-
plete when the protective groups were no longer detected by
1H NMR analysis. The methanolic solution of K2CO3 used here
removes benzyl and benzyloxycarbonyl groups simultaneously.
The reaction times for the deprotection reactions varied
between 4 and 8 h, and yields after purification ranged
between 87 and 91%. Comparative1H NMR spectra for PEG-b-
p(L-Glu100-co-L-Cys10) before and after the deprotection reac-
tions are shown in Figure 1. Protons indicative of the aro-
matic ring and methylene groups are no longer present in the
spectrum of the deprotected block copolymer.
coated with gold or gold nanoparticles were modified via: (i)
the terminal thiol group of the (L-Cys) repeat units in the PAA
block of PEGylated PAA block copolymers were used for a mul-
tipoint attachment of the block copolymers, see Scheme 2(a).
Here, the (L-Cys) building blocks formed either a random copol-
ymer with (L-Glu) repeat units, yielding PEG-b-p(L-Glu-co-L-Cys),
or a PAA diblock copolymer was formed, PEG-b-p(L-Glu)-b-p(L-
Cys). (ii) Surfaces were modified first by grafting the surfaces
with p(L-Lys)-SH and terminal amino groups in p(L-Lys) reacted
with polymeric aggregates carrying carboxyl groups in their
corona, HOOC-CH2-PEG-b-p(Bz-L-Glu)n, see Scheme 2(b). Here,
covalent amide linkages were formed between the polymeric
aggregate and the p(L-Lys) grafts present on the surface. This
method is complementary to the previously described chemi-
sorption between anionic surfaces and p(L-Lys)-g-PEG.40
Furthermore, diblock copolymers were synthesized using
HOOC-CH2-PEG-NH2 with a molecular weight of 5000 g
mol21 as macroinitiator for the polymerization of Bz-L-Glu-
NCA, and Bz-O-L-Lys-NCA, Table 1, Experiment 8, 9 yielding
HOOC-CH2-PEG-b-p(Bz-L-Glu)x and HOOC-CH2-PEG-b-p(Bz-O-
L-Cys)y. Because the glutamate and cysteine building blocks
in HOOC-CH2-PEGn-b-p(Bz-L-Glu)n, and HOOC-CH2-PEG-b-(Bz-
O-L-Cys)y are protected by benzyl groups and benzyloxycar-
bonyl groups, respectively, the copolymers are highly amphi-
philic and readily self-assemble in aqueous solution, forming
polymeric aggregates with a hydrodynamic radius of about
65 nm obtained by unweighted intensity data of DLS.28,39
As shown in Figure 2(a), a complete surface coverage was
achieved when PEG-b-p(L-Glu)100-b-p(L-Cys)20 was coated
onto the solid gold substrate. The surface layer is heterogene-
ous as a result of the phase separation of mutually immiscible
blocks of PEG and PAA. The higher magnification image of the
coating shown in Figure 2(b) reveals that in addition to the
phase separation on a 500-nm level, small spherical domains
in the size of a few nanometers are observed. It can be
assumed that this additional phase separation is the result of
the separation between the two PAA blocks. When the PAA
block exists as a random copolymer, only the phase separation
between the immiscible PEG and PAA blocks is observed, Fig-
ure 3. The small-scale separation as observed in Figure 2 is
not observed when PAA exists as p((L-Glu)100-co-(L-Cys)20).
When the molecular weight of the PAA block is reduced as in
PEG-b-p((L-Glu)50-co-(L-Cys)10) uniform surface films were
generated as well. The height images of Figure 3, clearly show
Block Copolymers on Solid Gold Surfaces
Two different concepts for the surface modification of solid sub-
strates were investigated. Three PEGylated PAA triblock copoly-
mers were selected, together with the p(L-Lys) grafted polymers
to be decorated with polymeric micelles. Specifically, substrates
a
spherical morphology of the phase separated block
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FIGURE 6 AFM phase image of (a) oligomeric p(L-Lys)15 attached covalently to a gold surface by the terminal thiol group and
(b) reactive micelles consisting of HOOC-CH2-PEG-b-p(Bz-L-Glu)13 covalently linked to the p(L-Lys) oligomeric surface layer.
copolymer. The more ordered phase separated morphology in
PEG-b-p((L-Glu)50-co-(L-Cys)10) is caused by the lower molecu-
lar weight of the PAA block as compared to the previously
discussed block copolymers (PEG-b-p(L-Glu)100-b-p(L-Cys)20).
this case is the result of the ability of p(L-Lys) to crystallize.
Reactive micelles consisting of HOOC-CH2-PEG114-b-p(Bz-L-
Glu)13 were covalently linked to the p(L-Lys) oligomers via
condensation between the terminal carboxyl groups of the
polymeric micelles and the amino functions on p(L-Lys).
These micelles cannot be washed off, indicating their cova-
lent attachment. The AFM image in Figure 6(b) shows
micelle sizes of approximately 50 nm, attached to p(L-Lys).
Block Copolymers on Gold Nanoparticles
Scheme 2(c) depicts the coating of Au nanoparticles. L-Cys
units adhere covalently to the nanoparticle. The PEG chains
phase-separate and crystallize on top of the nanoparticles.
Figure 4(a) shows that the block copolymer, here PEG-b-p((L-
Glu)50-co-p(L-Cys)10) bound selectively to the gold nanopar-
ticles, no polymer was deposited onto the intermittent silica
surfaces. In areas where the polymer is highly enriched,
larger polymer domains are formed that bridge the gold
nanoparticles. Because the PAA block is covalently attached
to gold nanoparticles, the PEG block must reside as the top
layer of the polymer coating, which can be seen in Figure
4(b). This morphology is clearly the result of the crystalliza-
tion of the PEG block and resembles screw dislocations.41
CONCLUSIONS
PEGylated PAA block copolymers were synthesized with a
narrow PDI, a good agreement between theoretical and
actual chain length was achieved. It was successfully demon-
strated that the thiol group of p(L-Cys) can be used to
anchor the block copolymers to solid gold surfaces and also
gold nanoparticles. The uniform coating of gold surfaces and
the bridging between gold nanoparticles leading to rather
uniform polymer layers indicate that anchoring PEGylated
PAA’s via L-Cys anchor sites is a viable approach to the coat-
ing of biomedically relevant surfaces.
If this polymer coating is exposed to an aqueous environ-
ment, as it would be in a biological or biomedical applica-
tion, the PEG block will coordinate with water and will form
polymer brushes that reach into the aqueous phase. The
WAXS data corroborate the results obtained by AFM as block
copolymers with large PAA blocks show only amorphous
halos for PEG-b-p(L-Glu)100-b-p(L-Cys)10 and PEG-b-p((L-
Glu)100-co-(L-Cys)20). However, when the molecular weight in
the PAA block is lower as in PEG-b-p((L-Glu)50-co-p(L-Cys)10),
PEG is able to crystallize as indicated by the two Bragg
reflections at 19.2 and 23.4ꢀ,42,43 see Figure 5.
ACKNOWLEDGEMENTS
This article was financially supported by NIH 1RO1EY016674-
04 and NIH ARRA 2RO1 EY016674-04A1, through a collabora-
tive effort with the Boston Retinal Implant Project. The authors
wish to thank Dr. Douglas. Shire of Cornell University, (Boston
Retinal Implant Project), Ithaca, NY for supplying gold coated
silicon wafers and Dr. Chengdu. Liang of Oak Ridge National
Laboratories, Oak Ridge, TN for producing the gold nanopar-
ticles on silicon wafers.
p(L-Lys)-Coated Surfaces Decorated with Polymeric
Micelles
Figure 6(a) shows the coating of a gold surface with oligo-
meric p(L-Lys), attached covalently to the gold surface by the
terminal thiol group, which is the result of initiating the
polymerization with cysteamine. The phase separation in
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