Thermosensitive peptide-hybrid ABC block copolymers obtained by ATRP: Synthesis, self-assembly, and enzymatic degradation

Article abstract of DOI:10.1021/ma2024667

Peptide-hybrid ABC block copolymers were synthesized by growing two different polymer chains from a native peptide using atom transfer radical polymerization (ATRP). To this end, two different ATRP initiators were coupled via orthogonal methods to the N- and C-terminus of the peptide Ser-Gly-Pro-Gln-Gly-Ile-Phe-Gly-Gln-Met-Gly, a substrate for matrix metalloproteases 2 and 9. First, a hydrophilic block of poly(oligo(ethylene glycol) methyl ether methacrylate) (pOEGMA) was polymerized from the peptide's C-terminus. Before polymerization of the second block, the first living chain end was inactivated by substitution of its Cl-terminus with azide under mild conditions. Then, a thermosensitive block of poly(N-isopropylacrylamide) (pNIPAm) was polymerized from the peptide's N-terminus. Well-defined polymers were obtained with good control over both block sizes. The resulting polymers self-assembled into micelles above the cloud point of the pNIPAm block. As anticipated, it was shown that the peptide linkage between the polymer blocks can be cut by a metalloprotease, leading to "shedding" of the corona of the micelles which makes these systems potentially suitable for enzyme-triggered drug delivery.

Full text of DOI:10.1021/ma2024667

Article
pubs.acs.org/Macromolecules
Thermosensitive Peptide-Hybrid ABC Block Copolymers Obtained by
ATRP: Synthesis, Self-Assembly, and Enzymatic Degradation
Albert J. de Graaf,† Enrico Mastrobattista,† Tina Vermonden,† Cornelus F. van Nostrum,†
Dirk T. S. Rijkers,‡ Rob M. J. Liskamp,‡ and Wim E. Hennink*,†
†Utrecht Institute for Pharmaceutical Sciences, Pharmaceutics, Utrecht University, P.O. Box 80.082, 3508TB Utrecht, The
Netherlands
‡Utrecht Institute for Pharmaceutical Sciences, Medicinal Chemistry & Chemical Biology, Utrecht University, P.O. Box 80.082,
3508TB Utrecht, The Netherlands
ABSTRACT: Peptide-hybrid ABC block copolymers were
synthesized by growing two different polymer chains from a
native peptide using atom transfer radical polymerization
(ATRP). To this end, two different ATRP initiators were
coupled via orthogonal methods to the N- and C-terminus of
the peptide Ser-Gly-Pro-Gln-Gly-Ile-Phe-Gly-Gln-Met-Gly, a
substrate for matrix metalloproteases 2 and 9. First, a
hydrophilic block of poly(oligo(ethylene glycol) methyl
ether methacrylate) (pOEGMA) was polymerized from the
peptide’s C-terminus. Before polymerization of the second
block, the first living chain end was inactivated by substitution of its Cl-terminus with azide under mild conditions. Then, a
thermosensitive block of poly(N-isopropylacrylamide) (pNIPAm) was polymerized from the peptide’s N-terminus. Well-defined
polymers were obtained with good control over both block sizes. The resulting polymers self-assembled into micelles above the
cloud point of the pNIPAm block. As anticipated, it was shown that the peptide linkage between the polymer blocks can be cut
by a metalloprotease, leading to “shedding” of the corona of the micelles which makes these systems potentially suitable for
enzyme-triggered drug delivery.
tion chain transfer (RAFT) polymerization,18−20 and nitroxide-
INTRODUCTION
The field of biohybrid materials (materials that consist of
■
mediated polymerization (NMP)21−23 allow for the synthesis of
low-dispersity polymers with defined, reactive chain-ends.24
A
synthetic materials together with biomolecules or even entire
cells) has been rapidly expanding over the past decade. This is
due to the promises it holds for pharmaceutical, medical, and
bio- and nanotechnological applications.1,2 Among others,
biohybrid materials are under investigation for use in targeted
drug delivery and for the controlled release of therapeutic
(bio)molecules. For example, biohybrid hydrogels can be used
as depot formulations of drugs or as scaffolds for tissue
engineering, whereas biohybrid micelles and vesicles are under
investigation for targeted drug delivery to inflamed tissue, e.g.,
in cancer and rheumatoid arthritis.3−8 In these materials, the
“bio” part may e.g. be a peptide containing a cell adhesion site
or an enzymatic cleavage site or a pH-, redox-, or temperature-
responsive peptide.4−6
A number of techniques are presently available for the design
of such peptide-hybrid polymers, including coupling a peptide
to a premade polymer (the “grafting to” approach),9,10
polymerizing a peptide-functionalized monomer (the “grafting
through” approach),11,12 or performing a polymerization using
a peptide macroinitiator (the “grafting from” approach).13,14
The advent of several controlled, “living” radical polymer-
ization techniques has greatly expanded the scope of these
“grafting” techniques. Techniques such as atom transfer radical
polymerization (ATRP),15−17 reversible addition−fragmenta-
variety of monomers can be polymerized using these
techniques, often under mild conditions. Furthermore, living
radical polymerizations are compatible with many functional
groups. Together, these properties render living radical
polymerizations suitable for use in “grafting from” and “grafting
through” strategies.25
In the present work we aimed at incorporating a peptide into
semisynthetic amphiphilic triblock copolymers, such that the
peptide is positioned between a hydrophilic and a thermo-
sensitive block. For the hydrophilic block of the final block
copolymer, oligo(ethylene glycol) methyl ether methacrylate
(OEGMA, Mn = 300 Da) was chosen as monomer. The
thermosensitive block was prepared by polymerization of N-
isopropylacrylamide (NIPAm). In aqueous solutions, these
polymers likely self-assemble into micellar structures above the
cloud point (CP) of the thermosensitive block as demonstrated
for other thermosensitive block copolymers.26,27 The peptide,
Ser-Gly-Pro-Gln-Gly-Ile-Phe-Gly-Gln-Met-Gly, was designed to
be functionalizable by specific chemistries on both its N- and C-
Received: November 8, 2011
Revised: December 26, 2011
Published: January 12, 2012
© 2012 American Chemical Society
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terminus. Furthermore, this peptide is cleavable at the Gly−Ile
bond by matrix metalloproteinases (MMPs) 2 and 9,28 which
are upregulated in inflamed tissues such as in cancer and
rheumatoid arthritis.29−31 Micelles, formed by self-assembly of
these amphiphilic block copolymers above the CP of the
pNIPAM block, can be loaded with a drug and be administered
intravenously. Because of the enhanced permeability and
retention (EPR) effect, they will accumulate in the inflamed
target tissue,32 where the hydrophilic stealth corona will
subsequently be selectively shed off by action of the MMPs.
The exposed micellar cores may then be taken up by the target
cells or aggregate and release their payload over time.33
Scheme 1. Synthesis Route of 2-(2-Aminoethoxy)ethyl-2-
bromoisobutyrate Hydrobromide (Linker 1)
In order to generate these thermosensitive block copolymers
with a peptide connecting the hydrophilic and thermosensitive
polymer blocks, in the present paper, two sequential polymer-
izations, each initiating on the other terminus of the same
peptide, were carried out. Thereby, full benefit is taken from
the advantages of the “grafting from” approach, mainly the high
coupling efficiency and easy work-up. To the best of our
knowledge, this is the first report of two sequential “grafting
from” polymerizations, initiated from the C- and N-terminus of
a peptide. Furthermore, we use the same polymerization
technique, ATRP, for both blocks. At present, sequential
“grafting from” has only been performed using a nonpeptide
initiator carrying two initiating moieties each for a different
polymerization chemistry.34,35 It is shown that well-defined
amphiphilic polymers can indeed be synthesized in this way.
These polymers self-assemble into micelles, having a corona
which can be shed off by action of a metalloprotease.
saturated NaCl. After drying on anhydrous MgSO4, the CH2Cl2 layer
was evaporated in vacuo, giving 2-(N-Boc-2-aminoethoxy)ethanol as a
1
colorless oil in 78% yield (15.9 g, 78 mmol). H NMR (300 MHz,
CDCl3): δ 5.28 (bs, 1H), 3.66 (t,2H), 3.50 (t, 2H), 3.48 (t, 2H), 3.25
(m, 2H), 3.10 (bs, 1H), 1.37 (s, 9H). 13C NMR (75.5 MHz, CDCl3):
δ 156.3 (CO), 79.4 (C(CH3)3), 72.3 (CH2), 70.3 (CH2), 61.5
(CH2), 40.4 (CH2), 28.4 (CH3).
In the next reaction step, 2-(N-Boc-2-aminoethoxy)ethanol (14.3 g,
70 mmol) and pyridine (6.2 mL, 77 mmol) were dissolved in
anhydrous CH2Cl2 (100 mL), and the solution was cooled on an ice
bath. To this solution, 2-bromoisobutyryl bromide (9.5 mL, 77 mmol)
was added dropwise while stirring. After addition, the mixture was
allowed to reach room temperature and stirred for 30 min. The
precipitated pyridinium bromide was filtered off. The filtrate was
washed three times with 1 M NaHSO4, three times with saturated
NaHCO3, and one time with saturated NaCl. After drying on
anhydrous MgSO4, the CH2Cl2 layer was evaporated in vacuo, giving 2-
(N-Boc-2-aminoethoxy)ethyl-2-bromoisobutyrate in 91% yield (24.4
g, 69 mmol).The product was a colorless oil which slowly crystallized
upon standing. 1H NMR (300 MHz, CDCl3): δ 4.93 (bs, 1H), 4.30 (t,
2H), 3.68 (t, 2H), 3.53 (t, 2H), 3.28 (m, 2H), 1.92 (s, 6H), 1.41 (s,
9H). 13C NMR (75.5 MHz, CDCl3): δ 171.8 (CO), 156.1 (CO),
79.4 (C(CH3)3), 70.3 (CH2), 68.6 (CH2), 65.0 (CH2), 55.7 (C−Br),
40.4 (CH2), 30.9 (C(CH3)2), 28.5 (C(CH3)3).
In the last reaction step, 2-(N-Boc-2-aminoethoxy)ethyl-2-bromoi-
sobutyrate (24 g, 68 mmol) was dissolved in diethyl ether (200 mL),
and HBr (28 mL of a 33% solution in acetic acid) was added dropwise,
after which the product precipitated out of solution. The reaction was
left at room temperature for 16 h. The resulting white crystals of 2-(2-
aminoethoxy)ethyl-2-bromoisobutyrate (1) were harvested by filtra-
tion, washed three times with a small volume of diethyl ether, and
dried in a vacuum. Yield: 16.1 g (48 mmol, 71%). 1H NMR (300 MHz,
CDCl3): δ 7.93 (bs, 3H), 4.39 (t, 2H), 3.89 (t, 2H), 3.81 (t, 2H), 3.32
(m, 2H), 1.94 (s, 6H). 13C NMR (75.5 MHz, CDCl3): δ 171.9 (C
O), 69.0 (CH2), 66.5 (CH2), 65.1 (CH2), 56.0 (C−Br), 40.0 (CH2),
30.9 (C(CH3)2). ESI-MS: calcd for C8H17NO3Br [M]+ 254.04; found
254.25.
EXPERIMENTAL PART
■
Chemicals. All solvents were obtained from Biosolve (Valkens-
waard, The Netherlands). Unless otherwise noted, the chemicals were
obtained from Sigma-Aldrich (Steinheim, Germany) and were used as
received. Prior to use, N,N-dimethylacetamide (DMA) was dried on
calcium hydride, distilled under reduced pressure, and stored over 4 Å
molecular sieves. Triethylamine was dried on potassium hydroxide,
distilled, and stored on 3 Å molecular sieves. Oligo(ethylene glycol)
methyl ether methacrylate with average Mn of 300 Da (OEGMA300
)
was passed over a column of basic alumina immediately prior to use.
Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was prepared and
purified according to a literature procedure.36
Analytical Methods. HPLC was performed on a Waters 2695
system equipped with a dual wavelength UV absorption detector set to
210 and 254 nm. An Alltech ProSpere C18 column was used at 25 °C,
employing a gradient of CH3CN/H2O/TFA 5/95/0.1 to 60/40/0.1 in
1 h. Unless noted otherwise, gel permation chromatography (GPC)
was performed on a Waters 2695 system equipped with a differential
refractive index detector using a MixedD column (Polymer
laboratories). The column temperature was 40 °C, 10 mM LiCl in
DMF was used as the eluent at a flow rate of 1 mL/min, and linear
PEG standards were used for calibration. Samples were allowed to
dissolve for at least 16 h prior to analysis.
NMR spectra were recorded on a Varian Mercury spectrometer
operating at 300 MHz (1H) or 75.5 MHz (13C), and ESI-MS spectra
were recorded on a Shimadzu QP8000 mass spectrometer. MALDI-
TOF MS was performed on a Kratos Axima CFR apparatus using
ACTH as external standard and α-cyano-4-hydroxycinnamic acid as
matrix.
Synthesis of 2-(2-Aminoethoxy)ethyl-2-bromoisobutyrate
Hydrobromide (Linker 1). The synthesis of linker 1 is shown in
Scheme 1. In a round-bottom flask, 2-(2-aminoethoxy)ethanol (11.0
mL, 110 mmol) and di-tert-butyl dicarbonate (21.8 g, 100 mmol) were
dissolved in CH2Cl2 (100 mL). The resulting mixture was stirred 16 h
at room temperature. Then, the mixture was washed three times with 1
M NaHSO4, three times with saturated NaHCO3, and once with
Synthesis of N-(3-Aminooxypropyl)-2-bromo-2-methylpro-
pionylamide Hydrochloride (Linker 2). The synthesis of linker 2 is
shown in Scheme 2. In a round-bottom flask, N-3-bromopropylph-
thalimide (24.5 g, 91 mmol) and N-Boc-hydroxylamine (13.3 g, 100
mmol) were dissolved in toluene (100 mL). The solution was boiled
under reflux, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (15 mL,
100 mmol) was added dropwise. During the addition of DBU the
desired product separated as a yellow oil. After stirring for 1 h, the
mixture was concentrated in vacuo. The residue was redissolved in
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1
−20 °C. Yield: 4.4 g (16 mmol, 89% over two steps). H NMR (300
MHz, DMSO-d6): δ 10.92 (bs, 3H), 8.18 (t, 1H), 4.01 (t, 2H), 3.17
(q, 2H), 1.87 (s, 6H), 1.75 (m, 2H). 13C NMR (75.5 MHz, DMSO-
d6): δ 170.8 (CO), 72.0 (CH2), 60.7 (C−Br), 35.9 (CH2), 31.1
(C(CH3)2), 27.2 (CH2). ESI-MS: calcd for C7H16N2O2Br ([M]+):
239.04; found 238.95.
Scheme 2. Synthesis Route of N-(3-Aminooxypropyl)-2-
bromo-2-methylpropionylamide Hydrochloride (Linker 2)
Peptide Synthesis. The peptide H-Ser-Gly-Pro-Gln-Gly-Ile-Phe-
Gly-Gln-Met-Gly-NH2 was synthesized by standard Fmoc solid phase
peptide synthesis.38 350 mg of peptide (TFA salt, 0.30 mmol) was
obtained, with a purity of >95% (HPLC). MALDI-TOF MS: calcd
1077.23 ([M + H]+); found 1077.21. The product was used without
further purification.
Functionalization of Peptide C-Terminus. To introduce a
homoserine lactone functionality at the C-terminus (Scheme 3),39−41
Scheme 3. Functionalization of the Peptide C-Terminus with
a
Linker 1
CH2Cl2 (200 mL) and washed with 10% citric acid (4 × 50 mL).37
The organic phase was dried over MgSO4 and concentrated in vacuo to
give N-(3-(N-Boc-aminooxy)propyl)phthalimide as a pale yellow solid
in 80% yield (23.5 g, 73 mmol). 1H NMR (300 MHz, CDCl3): δ 7.85
(t, 2H), 7.72 (t, 2H), 7.35 (s, 1H), 3.94 (t, 2H), 3.84 (t, 2H), 2.02 (m,
2H), 1.48 (s, 9H). 13C NMR (75.5 MHz, CDCl3): δ 168.5 (imide C
O), 157.0 (urethane CO), 134.1 (CH), 132.2 (C), 123.3 (CH),
81.8 (C(CH3)3), 74.0 (CH2), 35.1 (CH2), 28.3 (C(CH3)3), 27.3
(CH2).
In the next step, N-(3-(N-Boc-aminooxy)propyl)phthalimide (13 g,
40 mmol) was dissolved in absolute ethanol (50 mL). Hydrazine
hydrate (3 mL of an 85% aqueous solution) was added dropwise, and
the mixture was boiled under reflux for 2 h. The product separated as
its salt with phthalylhydrazide, from which it was liberated by adding
KOH (2.5 g dissolved in 20 mL of absolute ethanol). After stirring
vigorously for 1 h, the precipitate had turned into a fine white powder,
the potassium salt of phthalylhydrazide. The mixture was concentrated
in vacuo, resuspended in anhydrous CHCl3 (100 mL) under vigorous
stirring for 1 h, and filtered. The solids were washed three times with a
small amount of CHCl3. The combined filtrates were dried on MgSO4
and concentrated in vacuo to give 3-(N-Boc-aminooxy)propyl)amine
a
“Peptide” indicates the sequence H-Ser-Gly-Pro-Gln-Gly-Ile-Phe-Gly-
Gln-.
the peptide 3 (343 mg) was dissolved in 150 mL of N2-flushed
CH3CN/H2O/TFA (30/70/1). Under a nitrogen atmosphere, CNBr
(12 mL of a 4 M solution in CH3CN) was added, and the reaction
mixture was stirred for 16 h in the dark. The mixture was evaporated
to dryness in vacuo at 28 °C. The lactone-functionalized peptide 4 was
then redissolved in CH3CN/H2O/TFA (30/70/0.1) and lyophilized.
The yield after lyophilization was 383 mg. ESI-MS: calcd 973.47 ([M
+ H]+); found 973.47.
1
as an oil in quantitative yield (8 g, 40 mmol). H NMR (300 MHz,
Linker 1 (3.0 g, 0.9 mmol) was dissolved in CH2Cl2 (50 mL) and
converted into the free amine by shaking with an equimolar amount of
aqueous NaOH (9.0 mL, 1.0 M). The organic layer was dried on
MgSO4, followed by evaporation of the volatiles. Directly afterward,
1.2 mL of the resulting oil was added to the homoserine lactone-
functionalized peptide 4 (325 mg, 0.30 mmol) together with 2-
hydroxypyridine (200 μL of a 30 mg/mL solution in DMA). After
vigorous stirring for 15 min, the reaction mixture became
homogeneous, and it was then stirred for another 15 min. Excess
linker and DMA were removed by precipitation of the product in
MTBE (250 mL) containing 1% TFA. The resulting peptide
macroinitiator 5 was purified by preparative HPLC (SunFire C18
preparative column, gradient: CH3CN/H2O/TFA 30/70/0.1 to 50/
50/0.1, over 15 min) and subsequently lyophilized yielding 294 mg
(0.22 mmol) of the TFA salt of the pure peptide macroinitiator 5 as a
white powder. ESI-MS: calcd 1226.51 ([M + H]+); found 1226.30.
ATRP of the C-Terminal Polymer Block. Prior to use, all
solvents and liquid reagents were deoxygenated by flushing with
nitrogen gas for 15 min. A catalyst stock was prepared by weighing
CuCl (12.0 mg, 120 μmol), CuCl2·2H2O (13.6 mg, 80 μmol), and
2,2′-bipyridyl (bpy) (62.4 mg, 400 μmol) into a 7 mL glass screw-
CDCl3): δ 3.93 (t, 2H), 3.84 (t, 2H), 1.96 (bs, 2H), 1.75 (m, 2H),
1.46 (s, 9H). 13C NMR (75.5 MHz, CDCl3): δ 157.5 (CO), 82.0
(C(CH3)3), 75.0 (CH2), 39.1 (CH2), 29.4 (CH2), 28.4 (C(CH3)3).
ESI-MS: calcd for C8H19N2O3 [M + H]+: 191.14; found 190.90.
Subsequently, 3-(N-Boc-aminooxy)propyl)amine (6.9 g, 36 mmol)
and triethylamine (7.5 mL, 54 mmol) were dissolved in anhydrous
THF (70 mL). The mixture was purged with N2 and cooled on an ice
bath, after which 2-bromoisobutyryl bromide (6.7 mL, 54 mmol) was
added dropwise while stirring. Next, the ice bath was removed, and the
mixture was stirred for 1 h at room temperature. The precipitated
triethylammonium bromide was filtered off, and the filtrate was
concentrated in vacuo. The residue was redissolved in EtOAc (300
mL), and this solution was washed with 1 M NaHSO4 (3 × 50 mL), 1
M NaHCO3 (3 × 50 mL), and saturated NaCl (2 × 50 mL). The
organic layer was dried on MgSO4 and concentrated in vacuo to give
N-(3-(N′-Boc-aminooxy)propyl)-2-bromo-2-methylpropionylamide as
a yellow oil. A fraction (8 g) of this oil was dissolved in Et2O (25 mL),
and the solution was cooled on ice. HCl gas was bubbled through the
solution, upon which a white precipitate of N-(3-aminooxypropyl)-2-
bromo-2-methylpropionylamide hydrochloride (2) was formed. The
product was purified twice by trituration with CH2Cl2 and Et2O at
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capped vial equipped with a micro stirring bar. The vial was sealed
with a septum and flushed with nitrogen gas for 15 min. Then,
CH3CN (0.6 mL) and H2O (1.4 mL) were added through the septum,
and the vial was held in an ultrasonic bath until all solids had dissolved
forming the brown catalytic complex.
fluorescamine: 1 μL of sample was diluted in 1 mL of 50 mM
phosphate buffer of pH 8.9. Subsequently, to 150 μL of this solution
was added 50 μL of a freshly prepared 0.3 mg/mL solution of
fluorescamine in dry acetone. The fluorescence was recorded (λex
=
380 nm, λem = 460 nm) in a FluoStar Optima well plate reader (BMG
Labtech), and it was verified that after reaction with NaIO4 no
fluorescence was observable. To test for the development of an
aldehyde functionality, a drop of sample was placed on a TLC plate
and allowed to dry. The plate was then sprayed with a solution of 2.0 g
of 2,4-dinitrophenylhydrazine and 4.0 mL of concentrated sulfuric acid
in 100 mL of methanol. The development of a yellow spot indicated
the formation of an aldehyde 7.
After reaction with NaIO4, the solution was diluted to 10 mL in 50
mM anilinium acetate buffer, pH 4.6.43 Linker 2 (110 mg, 0.4 mmol)
was added, and the reaction was allowed to proceed for 16 h at room
temperature under nitrogen.
The polymer solution was again concentrated (4× 10-fold) using a
Vivaspin concentrator (MWCO 2, 5, and 10 kDa), exchanging the
buffer solution for demineralized water. The efficiency of coupling was
assessed by 1H NMR spectroscopy, comparing the integral of the
proton on the oxime carbon of 8, at 7.7 ppm, with the integral of the
aromatic phenylalanine protons.
ATRP of the N-Terminal Polymer Block. Following a published
procedure,44 CuBr (1.8 mg), CuBr2 (1.9 mg), and N-isopropylacry-
lamide (NIPAm) (320 or 640 mg for a target Mn of 16 and 32 kDa,
respectively) were weighed into a 2 mL septum vial. A stirring bar,
CH3CN (250 μL) and the N-terminally functionalized peptide-
pOEGMA 8 (1 mL of a 20 mM aqueous solution) were added. For the
target Mn of 32 kDa, it was necessary to add 1 extra mL of H2O and
250 μL of CH3CN to dissolve the NIPAm. The vial was placed in an
ice bath, and the solution was purged with N2 for 15 min. Then, the
reaction was started by adding 50 μL of a 0.4 M N2-purged aqueous
solution of Me6TREN. During the reaction, the conversion was
monitored by 1H NMR of samples diluted in air-saturated D2O.
Furthermore, at several time points samples were taken, which were
quenched by diluting in air-saturated DMF and analyzed by gel
permeation chromatography (GPC). The final polymers were dialyzed
against water and lyophilized.
Polymer Characterization. CPs of the polymers were determined
using a Shimadzu UV-2450 spectrophotometer with temperature
control by a Peltier element. The temperature of polymer solutions (1
mg/mL) was raised from 20 to 50 at 1 °C/min, and the CP was
defined as the onset of the curve of extinction at 650 nm vs
temperature.
For the determination of the critical micelle concentration (cmc),
the different block copolymers were dissolved in water in
concentrations ranging from 1 μg/mL to 1 mg/mL. Then, 5 μL of a
1.8 × 10−4 M solution of pyrene in acetone was added to 1 mL of
polymer solution. The micelles were formed by rapidly heating the
solutions to 40 °C. After incubation for 16 h at this temperature,
pyrene fluorescence was measured using a Horiba Fluorolog
fluorometer at 37 °C. The emission was measured at 390 nm using
excitation wavelengths of 333 and 338 nm. The ratio I338/I333 was
plotted against the logarithmic polymer concentration to determine
the cmc.45
OEGMA300 monomer (160, 320, or 640 μL for the aimed
pOEGMA block lengths of 4, 8, and 16 kDa, respectively) was
charged into an N2-filled 2 mL septum vial equipped with a stirring
bar. Then, 500 μL of the catalyst stock solution was added, followed by
the peptide macroinitiator 5 (40 μmol, dissolved in a minimal volume
1
of DMSO). Conversion was monitored during the reaction by H
NMR of samples diluted in air-saturated D2O, comparing the integral
of the region between 6.3 and 5.6 ppm (2H, H2CC) with the
integral of the region between 4.5 and 4.0 ppm (2H, C(O)OCH2).
Furthermore, at several time points samples were taken, which were
quenched by diluting in air-saturated DMF, and the evolution of
molecular weight was analyzed by GPC.
Azide Substitution of the Living Chain End. When the
monomer conversion was above 90%, NaN3 (80 μL of an N2-purged 1
M aqueous solution) was added to the reaction mixture, causing the
chloride chain end to be substituted by an azide by means of the
copper−bipyridyl catalyst.42 Since the azide-functionalized chain end
does not reinitiate, further OEGMA polymerization was prevented by
this procedure. Azide functionalization also allows conjugation with a
fluorescent probe.42 The reaction mixture was left for 16 h to ensure
complete substitution, after which the polymer was purified by 3× 10-
fold concentration using a Vivaspin concentrator (MWCO of 2, 5, or
10 kDa for pOEGMA block lengths of 4, 8, and 16 kDa, respectively),
each time diluting with 10 mM phosphate buffer, pH 7.0. The
1
molecular weight of the polymers was determined by H NMR based
upon the ratio of the integrals of the aromatic phenylalanine protons
and methoxy protons from pOEGMA.
Chain Extension Experiment. A sample (10 μL) of the peptide-
pOEGMA4
polymer solution was taken before and after the
kDa
substitution with NaN3. The samples were diluted with CH3CN (300
μL) and H2O (700 μL) in a 2 mL septum vial. Subsequently, NIPAm
(18 mg for target Mn = 32 kDa), CuBr (1.8 mg), and CuBr2 (1.9 mg)
were added. The vial was placed in an ice bath and purged with N2 for
15 min. Then, the reaction was started by adding Me6TREN (50 μL of
a 0.4 M N2-purged aqueous solution).
Functionalization of the N-Terminus. To introduce an aldehyde
functionality at the peptide’s N-terminus (Scheme 4), 60 μL of a 1 M
Scheme 4. Functionalization of the Peptide N-Terminus with
a
Linker 2
Formation of Micelles. Micelles were formed using a heat shock
procedure according to Neradovic et al.46 by heating 900 μL of 0.22
μm-filtered water or phosphate buffered saline (PBS) to 40 °C and
then adding 100 μL of a 1 mg/mL solution (at room temperature) of
the polymer. The mixture was kept at 40 °C for 5 min before being
equilibrated at 37 °C. Particle size was measured at 37 °C by dynamic
light scattering (DLS) on an ALV CGS-3 system at a 90° scattering
angle.
Fluorescent Labeling of the Polymer. To 1 mL of a 10 mg/mL
aqueous solution of pNIPAm32 kDa-peptide-pOEGMA8 kDa was added 3
μL of 0.1 mM copper(II) sulfate, 15 μL of 1 mg/mL Alexa Fluor 555
functionalized with an alkyne moiety in DMSO (Invitrogen), and 30
μL of 0.1 M ascorbic acid. The reaction mixture was purged with N2
and stirred for 16 h in an N2 atmosphere. The product was purified
using a Vivaspin membrane with a molecular weight cutoff of 10 kDa
a
“Peptide” indicates the sequence -Gly-Pro-Gln-Gly-Ile-Phe-Gly-Gln-.
aqueous solution of NaIO4 was added to the solution of peptide-
pOEGMA 6 (40 mM in 1 mL of 10 mM phosphate buffer pH 7.4).
Before and 15 min after addition, samples were taken for analysis.
Disappearance of the amino group was confirmed by reaction with
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Figure 1. ATRP of OEGMA300 using the synthesized peptide macroinitiator 5: (A) semilogarithmic plot of monomer concentration [M] (as [M]0/
■
▲
[M]) in time; (B) number-averaged molecular weight Mn (determined by GPC) as a function of monomer conversion. ( ) target Mn = 4 kDa, ( )
target Mn = 8 kDa, ( ) target Mn = 16 kDa.
▼
(3× 10-fold concentration, each time diluting with H2O). Labeling of
the polymer was verified by GPC using a MesoPore column (Polymer
Laboratories) at 40 °C, with DMF + 10 mM LiCl as the eluent and
both refractive index (RI) and fluorescence detection.
peptide with excess cyanogen bromide, 95% of the peptide was
converted into the lactone form 4 (HPLC). Apart from the
small amount of unreacted peptide, there was also a trace
impurity (<5%) of peptide in which the methionine residue had
been oxidized to methionine sulfoxide, which renders it
insensitive to lactonization with CNBr. The lactonized peptide
was used without purification, as the main impurities are not
reactive in the step of coupling the linker and could be removed
after that step.
Linker 1, which consists of a primary amine on one side and
an efficient 2-bromoisobutyrate ATRP initiator on the other
side, was synthesized in good yield and coupled to the peptide
(Scheme 3). To catalyze aminolysis of the lactone, 10 mol % of
2-hydroxypyridine was added.49 Nonreacted linker was easily
removed after the reaction with the peptide, by selective
precipitation, allowing the use of a large excess of linker. This
yields a very short reaction time and negligible inter- and
intramolecular reaction with the N-terminal amine. HPLC
showed that after 15 min ∼95% of the peptide lactone had
reacted. After preparative HPLC, the peptide macroinitiator 5
was obtained in 73% yield.
ATRP of OEGMA300 Starting from the C-Terminus. The
polymerization of OEGMA300 yields brush-shaped PEG-like
polymers which are frequently used as hydrophilic polymers in
the biomedical field since they possess several of the advantages
over the well-known PEG, e.g., the fact that they are
polymerizable by controlled radical polymerization.50−53
Furthermore, because of the hydrophilic oligo(ethylene glycol)
side chains and the hydrophobic polymethacrylate backbone,
pOEGMA’s have thermosensitive properties, which can be
tuned by the average length of the oligo(ethylene glycol) side
chains.53 By using a monomer with an average molar mass of
300 Da, it was ensured that the resulting polymer has a lower
critical solution temperature (LCST) above 68 °C,52 meaning
that the polymer is water-soluble at physiological temperature.
For the polymerization, a mild method compatible with the
peptide was developed. Generally, ATRP of OEGMA is
performed in alcohols at elevated temperatures,51,53 but it can
also be performed in aqueous media at room temperature.50 By
addition of Cu(II) and a small amount of CH3CN, a
pseudoligand for Cu(I), the problem of disproportionation
commonly faced in aqueous ATRP was eliminated.54,55
Furthermore, by varying the percentage CH3CN (and
consequently the polarity of the solvent), it is possible to
adjust the rate of the reaction.56 In the present study, a fast
reaction with sufficient control at ambient temperature was
obtained in a CH3CN/H2O 3/7 (v/v) solvent mixture, which is
Enzymatic Degradation. Metalloprotease (type IV collagenase)
from C. Histolyticum was used as a model for MMP-2 and MMP-9.
This enzyme has very similar substrate specificity and is commonly
used as a readily available alternative to MMP-2 and MMP-9.3,4 The
activity of the enzyme was determined colorimetrically by the N-(3-[2-
furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) cleavage assay according
to the manufacturer’s protocol (Sigma-Aldrich) and was found to be
2300 units/mg (1 unit represents an activity of 1000 pmol substrate/
min). The fluorescently labeled polymer was diluted to a final polymer
concentration of 10 μM in 0.22 μm-filtered HEPES buffer of pH 7.4
containing 20 mM CaCl2 and 100 mM NaCl at room temperature.
Then, a solution (0.22 μm-filtered) of 1000 units/mL of type IV
collagenase in the same buffer was added to a final enzyme
concentration of 10 units/mL, and the solution was incubated for
24 h at room temperature. GPC analysis with fluorescence detection
was performed before and after the degradation (MesoPore column at
40 °C, DMF + 10 mM LiCl).
For the enzymatic degradation of intact micelles, micelles were
formed by the above-mentioned heat shock procedure in HEPES
buffer after which they were incubated with collagenase at 37 °C. At t
= 0 and after 24 h a sample was diluted 10-fold in preheated buffer and
injected into a Nanosight LM10-HS laser light scattering/fluorescence
microscopy system, preheated to 37 °C. Using the Nanoparticle
Tracking Analysis (NTA) software, images were taken to visualize the
micelles and to determine whether their fluorescently labeled coronas
had been cleaved off.
RESULTS AND DISCUSSION
■
Functionalization of the Peptide C-Terminus. To
activate selectively the C-terminus of the peptide 3, a reactive
homoserine lactone was introduced by reaction of the
penultimate methionine residue with CNBr (Scheme 3). This
method, which is commonly used for the cleavage of
recombinantly produced peptides from a carrier fusion
protein,41 allows for rapid and selective modification of the
C-terminus of any peptide having methionine as the
penultimate residue on the C-terminus.39,40 The only require-
ment is the absence of internal methionine residues, which
would lead to cleavage of the peptide. However, since
methionine is one of the least occurring amino acids in
proteins,47 this requirement is often easy to meet, making
methionine a favorable target for site-specific peptide
conjugation. The main advantage over other methodologies
that target the C-terminus, e.g., carbodiimide-based coupling,48
is that CNBr functionalization is compatible with the presence
of glutamate and aspartate residues. After reaction of the
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a good solvent for the peptide used in the present study. As can
be seen in Figure 1A, the residual monomer concentration
decreased exponentially in time during the course of the
reaction. This indicates a constant concentration of propagating
radicals during the polymerization and thus effective and
instantaneous initiation as well as negligible termination/
combination; both are prerequisites for a controlled/living
polymerization.57 Furthermore, the number-averaged molecular
weight (Mn) evolved linearly with conversion (Figure 1B),
indicating a low rate of termination. These observations
indicate that the polymerization was controlled.
phosphate buffer (pH 7.4) (Scheme 4). Reaction with
fluorescamine indicated disappearance of the amine function-
ality of the N-terminal serine. Furthermore, a sample taken
from the reaction mixture developed a yellow color after
addition of 2,4-dinitrophenylhydrazine, indicating the forma-
tion of an aldehyde.
For further functionalization of the aldehyde-modified
terminus of the peptide 7, linker 2 was developed, carrying
on one side a reactive aminooxy group and on the other side an
ATRP initiator functionality. Linker 2 was synthesized as
shown in Scheme 2. Subsequently, the aldehyde-functionalized
peptide-pOEGMA conjugates 7 were incubated with linker 2 in
50 mM anilinium acetate buffer (pH 4.6) (Scheme 4), a known
catalyst for the formation of oxime bonds.71,73,74 After coupling
of linker 2, the appearance of a peak at δ 7.7 ppm in 1H NMR
(in D2O) indicated the formation of an oxime bond (Figure 3).
1H NMR spectroscopy of the polymers after purification
showed that the pOEGMA blocks had Mn values of 3.2, 7.1,
and 15.7 kDa, which are in good agreement with the expected
molecular weights.
Inactivation of the Living Chain-End. One feature of
ATRP is that the living chain end of the resulting polymers is
able to reinitiate another polymerization, and this feature has
been frequently used for the synthesis of block copoly-
mers.58−61 To be able to grow a different polymer chain
from the other (N-) terminus of the peptide, however, it must
be ensured that the already existing polymer chain on the C-
terminus will not reinitiate in a subsequent ATRP. Therefore,
the chloride on the living chain end was substituted by azide
using a recently developed copper-catalyzed azide substitution
reaction.42 The introduced azide functionality also renders it
possible to attach in a later stage a fluorescent probe or a
targeting ligand using azide/alkyne “click” chemistry.42,62−68
Figure 2 shows that the substitution with NaN3, as expected,
Figure 3. NMR spectrum of linker 2-peptide-pOEGMA4 kDa showing
the peak of the proton on the oxime carbon at δ 7.7 ppm and the
aromatic phenylalanine protons at δ 7.3−7.4 ppm.
The degree of functionalization was >90% based on
comparison of the integral of this peak with the integral of
the peak of the phenyl protons of phenylalanine (δ 7.5−7.2
ppm).
ATRP of the N-Terminal Block. NIPAm was polymerized
from the modified N-terminus of the peptide-pOEGMA
conjugates 8 by ATRP in a CH3CN/H2O 3/7 (v/v) mixture
at 0 °C.44,56,75 The reaction was well controlled as evidenced by
an exponential decrease of the residual monomer concentration
(Figure 4A) and a linear evolution of Mn with conversion, for
both target molecular weights of the pNIPAm block (Figure
4B). This indicates successful growth of the pNIPAm block,
starting from the peptide-pOEGMA macroinitiator 8.
Characterization of the Polymers and Their Micelles.
Figure 2. GPC chromatograms of peptide-pOEGMA4
hybrid
kDa
polymer: (a) before substitution of the chloride chain end with azide,
(b) after substitution with azide, (c) non-azidated polymer after chain
extension with NIPAm, and (d) azidated polymer after chain
extension.
did not alter the molecular weight (distribution) of the polymer
as determined by GPC analysis. Furthermore, the non-
substituted pOEGMA chain could be extended with a pNIPAm
block, which provides indeed evidence for the living character
of the ATRP of OEGMA on the peptide macroinitiator. On the
other hand, after substitution with NaN3, pOEGMA was not
able to reinitiate an ATRP (Figure 2). This demonstrates
quantitative substitution of the Cl end-group by azide.
Functionalization of the N-Terminus. Introduction of an
aldehyde functionality into a peptide by mild periodate
oxidation of an N-terminal serine residue followed by coupling
of an O-substituted hydroxylamine is a highly selective,
efficient, and well-known bioconjugation reaction.69−71 The
formed oxime bonds are acid-sensitive but stable at
physiological pH.72 The peptide-pOEGMA conjugates 6 were
subjected to the above-mentioned oxidation with NaIO4 in
1
The structure of the final polymers is depicted in Figure 5. H
NMR analysis showed that the pNIPAm blocks had Mn values
in good agreement with the expected molecular weight, based
on the comparison of the integrals of the pOEGMA methoxy
protons and pNIPAm methyl protons (Table 1). Furthermore,
the dispersity of the synthesized polymers was low, with only
polymers containing a pOEGMA16 kDa block having a somewhat
broader size distribution.
The cmc of the polymers dissolved in water was equal for all
polymers. In general, a decrease in cmc with increasing
hydrophobic block length would be expected, as well as an
increase in cmc with increasing hydrophilic block length.76,77
Our observation of equal cmc's might be related to the fact that
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Figure 4. Kinetics of the polymerization of NIPAm onto peptide-pOEGMA8 kDa: (A) semilogarithmic plot of monomer concentration [M] (as
■
▲
[M]0/[M]) in time; (B) number-averaged molecular weight Mn as a function of monomer conversion. ( ) Target Mn = 16 kDa, ( ) target Mn = 32
kDa.
Figure 5. Structure of the final biohybrid triblock polymers. The wavy line indicates the bond that is cleavable by MMPs. The asterisk indicates the
attachment point of the fluorescent probe in the P8N32 polymer.
Table 1. Properties of the Polymers and Their Micelles
polymers
micelles
b
d
f
Mn (kDa)
CP (°C)
Rh (nm) (PDI)
a
c
e
abbrev
P4
pOEGMA
pNIPAm
Đ
H2O
PBS
cmc (mg/mL)
H2O
PBS
3.2
3.2
1.20
1.25
1.24
1.32
1.24
1.26
1.45
1.52
1.64
P4N16
P4N32
P8
16.0
31.2
35.5 0.1
34.5 0.1
33.0 0.2
32.0 0.2
0.03 0.01
0.03 0.01
29 1 (0.1)
28 1 (0.1)
173 2 (0.1)
3.2
1756 138 (0.5)
7.1
P8N16
P8N32
P16
7.1
17.0
30.8
35.6 0.1
34.8 0.1
33.1 0.1
32.2 0.1
0.03 0.01
0.03 0.01
27 1 (0.1)
27 1 (0.1)
35 1 (0.1)
123 2 (0.1)
7.1
15.7
15.7
15.7
P16N16
P16N32
15.6
33.3
36.0 0.1
34.7 0.1
33.2 0.1
32.1 0.1
0.03 0.01
23 1 (0.2)
22 1 (0.1)
24 1 (0.1)
33 1 (0.1)
0.03 0.01
a
b
P denotes the aimed size of the pOEGMA block and N that of the pNIPAm block (both in kDa). Number-averaged molecular weight based on 1H
c
d
e
f
NMR. Dispersity from GPC. Cloud point. Critical micelle concentration. Z-averaged hydrodynamic radius (Rh) from DLS.
the found cmc values are very low, permitting an influence from
the fluorescent probe (pyrene) itself.78
The CPs of the polymers dissolved in water were slightly
higher than the published value of 32 °C of pNIPAm
homopolymer in water due to the presence of large hydrophilic
polymer blocks.79,80
Consequently, Table 1 shows that the CP in water increased
from 35.5 to 36.0 °C with increasing the pOEGMA block
length from 4 to 16 kDa (P4N16 vs P16N16) and decreased
with increasing the pNIPAm length from 16 to 32 kDa. For
self-assembly into micelles, a heat-shock protocol was used as it
has been described that instantaneous heating of aqueous
polymer solutions to above their CP leads to well-defined
micelles.46 Following this procedure, all polymers formed
micelles with a size of 22−29 nm in water and narrow size
distributions, which is favorable for drug delivery purposes.81
Increasing the pOEGMA length from 4 to 16 kDa leads to a
larger surface area that is needed per polymer chain and thus to
less polymer chains fitting in one micelle:44 consequently, the
size of the micelles decreased from 29 nm (P4N16) to 23 nm
(P16N16). Interestingly, increasing the pNIPAm block from 16
to 32 kDa did not lead to an increase of the micellar size, but to
a small decrease, e.g., 29 nm (P4N16) vs 28 nm (P4N32). This
effect has been observed before and has been attributed to
greater hydrophobicity of the longer pNIPAm blocks, leading
to more extensive dehydration of the micellar cores.46,82
The difference in CP and size between micelles dispersed in
water and PBS is also striking. The salting-out effect of PBS
reduced the CPs by 2−3 °C, to values (32−33 °C) that are well
below physiological temperature and thus compatible with drug
delivery applications. Furthermore, PBS also led to a higher
observed Z-averaged size. PEG is known to partially dehydrate
upon addition of salt,46,83,84 and the same behavior may be
expected for pOEGMA. This partial dehydration may lead to
the formation of larger particles due to a change in the ratio
between the hydrodynamic volumes of the hydrophilic and
hydrophobic blocks. For large pOEGMA blocks and/or small
pNIPAM blocks, the hydrophilic/hydrophobic ratio in buffer is
still enough to form small micelles (P8N16, P16N16, and
P16N32); for smaller pOEGMA blocks, the increase in size was
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more pronounced. For the P4N32 polymer, the (decreased)
hydrodynamic volume of the pOEGMA blocks in buffer was
not enough anymore to support stable nanoparticles, and
aggregation resulted.
Enzymatic Degradation. The peptide separating the
hydrophilic micelle corona from the thermosensitive micelle
core has been designed to be cleaved by MMP-2 and MMP-9,
as these enzymes are upregulated in diseased tissues (e.g., in
cancer and rheumatoid arthritis). For this reason, MMP-2/
MMP-9 substrates have previously been utilized as building
blocks of tissue-specific drug delivery systems.85−90
light or by their fluorescence. Images were obtained in both
laser scattering and fluorescence mode directly before addition
of the enzyme and after 24 h incubation at 37 °C (Figure 7). As
can be seen in Figure 7C, particles are still present after
degradation, but their fluorescence has vanished into the
To verify that the peptide could still be cleaved by
metalloproteases after growing the two polymer blocks on its
N- and C-termini, the P8N32 block copolymer was
fluorescently labeled at the end of its hydrophilic block.
Alkyne-functionalized Alexa Fluor 555 was coupled (“clicked”)
to the azide-functionalized chain end by Cu(I)-catalyzed azide−
alkyne cycloaddition. A label to polymer molar ratio of 1:10 was
used in order to minimize any effects of the label on micelle
formation. As shown in Figure 6, the polymer was successfully
labeled with Alexa Fluor 555 by this procedure.
Subsequently, the polymer was incubated with collagenase
for 24 h at room temperature. The cleaved polymer was then
Figure 7. Images of the micellar dispersion of fluorescently labeled
P8N32 polymer, taken using a Nanosight system before (A and B) and
after (C and D) enzymatic cleavage in HEPES buffer. (A) and (C)
were recorded in scattering mode and (B) and (D) in fluorescence
mode (λex = 532 nm, λem > 565 nm), keeping the camera gain settings
constant. Scale bars correspond to 10 μm; however, the apparent size
of the micelles in these images reflects their scattering intensity rather
than their actual size.
background noise (Figure 7D), indicating that the fluorescent
and highly mobile hydrophilic pOEGMA chains have been
cleaved off by the enzyme. On the basis of these findings, it can
be expected that when these micelles are used for drug delivery
to tumors or inflamed tissues, their “stealth” corona will be
cleaved off at the target site of action. We hypothesize that this
“shedding” of the corona will impede further circulation of the
micelles and facilitate cellular uptake.
Figure 6. GPC traces of the fluorescently labeled polymer: (a) RI
signal after labeling, (b) fluorescence signal (λex = 555 nm, λem = 585
nm) after labeling, (c) fluorescence signal after enzymatic cleavage. For
comparison, (d) shows the RI signal of peptide-pOEGMA8 kDa
.
analyzed by GPC with fluorescence detection. It is well-known
that pNIPAm (co)polymers elute in GPC at retention times
corresponding to much higher molecular weights than
expected, probably due to very persistent interchain hydrogen
bonds.91,92 This feature was exploited for the demonstration of
cleavage of the peptide−polymer conjugate by collagenase.
When a column with a narrow separation range was used, the
fluorescently labeled intact polymer elutes in the void volume
(at 14 min, Figure 6), whereas the fluorescently labeled
pOEGMA that is cleaved off elutes much later (at 18 min, the
same retention time as peptide-pOEGMA8 kDa). Since the
pNIPAm is unlabeled after cleavage, it is invisible using
fluorescence detection. Thus, this method leads to a good
separation of the uncleaved polymer and the cleaved-off
hydrophilic blocks, while preventing interference from the
cleaved thermosensitive blocks which coelute with the
uncleaved polymer but are invisible using fluorescence
detection.
CONCLUSION
■
We have demonstrated a suitable approach to grow two
different polymer chains from a native peptide by ATRP, using
two orthogonal methods to couple ATRP initiators to the N-
and C-terminus. Furthermore, in this work a mild method is
presented to inactivate the first living ATRP chain end, allowing
the same polymerization chemistry for both polymerizations.
Both polymerizations were well controlled, leading to a well-
defined end product with control over the desired polymer
block lengths.
Above the cloud point of one of the blocks, the polymers
self-assembled into micelles. The micelles have been shown to
“shed” the hydrophilic polymer blocks on their outside by the
action of collagenase, a model for diseased tissue-specific matrix
metalloproteases. Thus, the technology presented herein offers
new possibilities for enzyme-triggered drug delivery.
To visualize the enzymatic degradation of the particles,
micelles were formed by heat-shocking the fluorescently labeled
polymer. Images of the solution were taken using a preheated
Nanosight LM10-HS microscopy system which allows visual-
ization of the nanoparticles by either their scattering of laser
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: w.e.hennink@uu.nl.
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