Reactive thermoresponsive copolymer scaffolds

Article abstract of DOI:10.1039/c0cc03856c

Thermoresponsive copolymer scaffolds containing reactive aldehyde functions were prepared and a selection of organic residues conjugated to these copolymer scaffolds through oxime/hydrazone formation. The conjugation of hydrophobic residues affords copoly

Full text of DOI:10.1039/c0cc03856c

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Reactive thermoresponsive copolymer scaffoldsw
Benjamin S. Murray, Alexander W. Jackson, Clare S. Mahon and David A. Fulton*
Received 14th September 2010, Accepted 24th September 2010
DOI: 10.1039/c0cc03856c
Thermoresponsive copolymer scaffolds containing reactive
aldehyde functions were prepared and a selection of organic
residues conjugated to these copolymer scaffolds through oxime/
hydrazone formation. The conjugation of hydrophobic residues
affords copolymers whose lower solution critical temperatures
are in most cases higher than that of the parent copolymer
scaffold.
well-known thermoresponsive polymer—as there are no
toxicity issues yet their thermosensitivity is almost independent
of external conditions. In particular, we chose to focus on
polymers derived from the OEGMA300 monomer which possesses
a LCST of B60 1C.5 The known aldehyde methacrylate
p-(methacryloxyethoxy)benzaldehyde6 (MAEBA) was chosen
as a suitable monomer to incorporate reactive aromatic
aldehyde functional groups into the polymer chain. Aldehydes
are an attractive functional group to facilitate polymer
functionalization through hydrolytically stable oxime or
hydrazone bond formation with readily available nitrogen
nucleophiles.7 To ensure accurate comparisons between different
polymers it is vitally important that they possess uniform
molecular weights, and for this reason we have utilized living
radical polymerization techniques to prepare our polymer
samples. Reversible addition–fragmentation chain transfer
(RAFT) polymerization8 is particularly appealing because of
its versatility and experimental simplicity, and has previously
demonstrated great utility in polymerizing OEGMA monomers
with a high level of control.9 The RAFT chain transfer agent
4-(4-cyanopentanoic acid)dithiobenzoate (CPADB) was used
to mediate the copolymerization of OEGMA300 and MAEBA
(Scheme 1) in dioxane at 70 1C to produce a series of
copolymers P1–P6 of similar lengths but possessing different
ratios of the two monomers. These copolymers, which display
excellent water-solubility, were characterized (Table 1) by gel
permeation chromatography using THF as eluant, displaying
monomodal distributions in all cases and polydispersity
Thermoresponsive polymers are an important and extremely
useful class of macromolecule which display great promise in
numerous nanotechnological and biomedical applications.1
These polymers undergo a reversible phase transition from
soluble to insoluble when their solution temperature is
raised above their lower critical solution temperature (LCST),
changing from an extended chain conformation below this
temperature into a collapsed chain above this temperature.
The utility of thermoresponsive polymers could be increased
if they possessed reactive functional groups within their side
chains to allow the conjugation of other molecules e.g. peptides,
carbohydrates, targeting vectors or drugs, onto the polymer
scaffold. There are, however, remarkably few examples
described in the literature of thermoresponsive polymers
featuring reactive functional groups.2 The groups of Brooks
and Kizhakkedathu have prepared2a poly(N-[(2,2-dimethyl-
1,3-dioxolane)methyl]acrylamide), which can be transformed
through cleavage of the pendant dioxolane groups followed by
oxidation of the resultant diol to afford a thermoresponsive
polymer scaffold possessing reactive aldehyde groups.
Although this work represents a significant advance, there still
remains a need for a thermoresponsive polymer scaffold which
can be readily prepared and requires no post-polymerization
transformations to activate the reactive functional groups. We
report here a thermoresponsive random copolymer scaffold
displaying reactive aldehyde groups on its side chains and
demonstrate that examples of alkoxyamine and hydrazide
residues can be successfully conjugated onto the scaffold,
subsequently influencing the copolymers LCST.
We chose to base our copolymer scaffold upon poly-
oligo(ethylene glycol) methacrylate polymers, a relatively
new class of thermoresponsive polymer possessing a high
degree of biocompatibility and tunable LCSTs.3 These non-
linear poly(ethylene glycol) (PEG) analogues are alternative
thermoresponsive polymers to poly-N-isopropylacrylamide4—a
Chemical Nanoscience Laboratory, School of Chemistry,
Bedson Building, Newcastle University, Newcastle upon Tyne, UK.
E-mail: d.a.fulton@ncl.ac.uk; Fax: +44 (0)191 222 6929;
Tel: +44 (0)191 222 7065
w Electronic supplementary information (ESI) available: All experi-
mental procedures and characterization of all polymers (P1–P6),
conjugation procedures and turbimetric analysis of all conjugates.
See DOI: 10.1039/c0cc03856c
Scheme 1 RAFT polymerization of OEGMA300 with methacrylate
p-(methacryloxyethoxy)benzaldehyde (MAEBA) to form reactive
thermoresponsive copolymer scaffolds.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8651–8653 8651

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Table 1 Characterization of polymers P1–P6
Comonomer ratio
OEGMA300 : MAEBAa
Polymer
Molar wt% of MAEBA
Mnb/g molÀ1
Mwb/g molÀ1
PDI
LCSTc/1C
P1
P2
P3
P4
P5
P6
2 : 1
3 : 1
4 : 1
8 : 1
28.0%
20.5%
16.5%
9.0%
4.5%
0%
13 700
13 000
13 800
12 200
15 800
17 700
18 500
18 300
19 400
16 400
21 800
25 000
1.35
1.41
1.40
1.34
1.38
1.41
22.0
31.0
39.0
49.0
57.5
66.0
16 : 1
OEGMA300 homopolymer
a
b
c
As determined by 1H NMR spectroscopy. As determined by gel permeation chromatography in THF. As determined by turbimetric analysis
in 0.1 M NaCl solution at pH 7.4. LCST defined as the onset of sharp change in transmission at 550 nm.
Fig.
1 Temperature–turbidity curves for copolymers/polymers
P1–P6 measured in 0.1 M NaCl solution at pH 7.4.
Fig. 2 1H NMR spectrum (400 MHz, D2O) before (A) and after (B)
conjugation of residue 2 onto copolymer P3 (R = CH2CO2H).
indices 1.34–1.41, and by 1H NMR spectroscopy. The monomer
1
composition of each copolymer was determined by H NMR
is shown (Fig. 2). As can be observed, the aldehyde signal at
d = 9.8 ppm (Fig. 2A) is greatly decreased in intensity after
the conjugation (Fig. 2B) whilst signals for cis/trans-oxime are
observed at d = 8.0 and 8.3 ppm, confirming efficient
conjugation.
spectroscopy and found to have an excellent match with the
theoretical composition as determined by the monomer feed
ratios.
The LCSTs of P1–P6 were measured by turbimetric analysis
(Table 1, Fig. 1) in pH controlled aqueous solution, indicating
that as expected the LCST decreases linearly as the ratio of the
more hydrophobic monomer MAEBA within the copolymer
increases (see ESIw, Fig. 1), demonstrating that the LCST of
these copolymers can be tuned by varying the comonomer
composition. Further studies indicated that the LCSTs of
copolymers P1–P6 was also found to be dependent on salt
concentration and pH. The presence of 0.1 M NaCl (pH 7.4)
resulted in a reduction in the LCST of B4 1C compared to
identical samples in purite water (pH 7.4) for all polymers at a
concentration of 2 mg mLÀ1. This observation can be explained
by the partial dehydration of the PEG side-chains in the
presence of NaCl as reported previously.10 Similarly, a change
in pH from 4.0 to 7.4 in a 0.1 M NaCl background resulted in
an increase in LCST of B3–4 1C, observations which can be
attributed to the deprotonation of the carboxylic acid end
group of the polymers, increasing their hydrophilicities.11
To investigate the effectiveness of these copolymers as
reactive scaffolds, a selection of small molecule alkoxyamine
and hydrazide residues were conjugated (see ESIw) onto the
polymer scaffolds through oxime or hydrazone bonds. The
selection of residues was chosen to include hydrophilic residues
(1a and b), an ionic residue (2) and hydrophobic residues (3–8).
For the residues studied, the conjugations were found to be
highly efficient in every case. A typical 1H NMR spectrum for
the conjugation of O-(carboxymethyl)hydroxylamine 2 in D2O
The LCSTs of a selection of conjugated copolymers
were measured (Table 2) by turbimetric analysis (see ESIw).
Conjugation of the hydrophilic tetraethylene glycol residue 1a
to the copolymer scaffolds P1, P3 and P4 possessing ratios of
monomers OEGMA300 : MAEBA of 2 : 1, 4 : 1 and 8 : 1,
respectively, were found to afford LCSTs corresponding to
increases of 20.0 1C, 9.5 1C and 8.5 1C, respectively, relative to
the parent copolymer scaffolds. Conjugation of the shorter
Table 2 Measured LCST values for the conjugation residues 1–8
onto copolymer P3, and their differences relative to P3 (final entry)
under identical conditions
Residue
LCSTa/1C
DT relative to P3/1C
Dhb/nm
1a
1b
2
3
4
5
6
7
8
P3
48.5
42.5
—
44.0
43.0
43.0
42.0
42.0
38.0
39.0
9.5
3.5
—
5.0
4.0
4.0
3.0
3.0
À1.0
—
7.5
6.7
7.6
8.8
8.4
8.9
10.0
9.8
8.5
6.1
a
As determined by turbimetric analysis in 0.1 M NaCl solution at
pH 7.4. LCST defined as the onset of sharp change in transmission.
b
As determined by dynamic light scattering.
c
8652 Chem. Commun., 2010, 46, 8651–8653
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hydrophilic residue 1b to the copolymer scaffold P3 afforded a
conjugate whose LCST is only 3.5 1C higher than P3. These
observations are expected as the conjugation of hydrophilic
residues such as 1a–b should increase the hydrophilicity of the
copolymers and consequently raise their LCSTs. The copolymer
conjugates derived from conjugation of the hydrophobic
residues 3–8 onto copolymer scaffolds P3 were found to
exhibit LCSTs either greater or comparable to that of the
parent copolymer, observations which were unexpected.12
The measured hydrodynamic diameters (Table 2) of these
‘hydrophobic’ conjugated copolymers below their LCSTs
indicate they exist as unimers in solution, suggesting that
the unexpected increase in LCST is not as a consequence of
the formation of micellar aggregates. The reasons for the
unexpected LCSTs of the ‘hydrophobic’ conjugated copolymers
are not clear and an in-depth study beyond the scope of
this paper, but may involve the disruption by hydrophobic
appendages of the interfacial waters associated with the
polymer chain, resulting in an increase in entropy of the
polymer (see ESIw for an in-depth discussion). An implication
of these observations is that it suggests poly-oligo(ethylene
glycol) methacrylate-based polymer scaffolds can be ‘loaded’
with hydrophobic residues without losing their thermoresponsive
properties, a feature which could be important in fields such as
drug delivery.
In conclusion, we have described here thermoresponsive
copolymer scaffolds bearing reactive aldehyde functionalities
which can be conjugated with small molecule residues through
oxime formation. Advantages of this copolymer are: (i) it is
easy to prepare and does not require any deprotection or
post-polymerization modification steps to make the aldehyde
function available for reaction, (ii) it is fully water soluble and
principally composed of biocompatible oligo(ethylene glycol)
segments, (iii) nitrogen nucleophiles such as alkoxyamines and
hydrazides can conjugate onto the copolymer with high yields
through formation of hydrolytically stable carbon–nitrogen
double bonds, and (v) the LCST of the copolymer scaffold can
be tuned by changing the composition of the residues
conjugated to the scaffold. Furthermore, by using a controlled
living radical polymerization method such as RAFT, the
structural properties of the polymer i.e. the length of the
polymer, the density of aldehyde groups, the type of OEGMA
monomers used, can easily be tuned thus allowing quick access
to a series of polymer scaffolds. The unexpected observation
that the hydrophobic residues can, in most cases studied, be
conjugated to these copolymer scaffolds whilst maintaining or
increasing the LCST of the resulting conjugates may also
increase the potential utility of these copolymer scaffolds.
We wish to thank EPSRC for support. We also thank the
regional development agency One North East for considerable
support. CSM thanks The Nuffield Foundation for the award
of an undergraduate research science bursary.
Notes and references
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The conjugation of the ionic residue O-(carboxymethyl)-
hydroxylamine (2) to copolymer scaffold P3 in 0.1 M NaCl
solution afforded a copolymer conjugate whose LCST was
strongly pH dependent. At pH 3.8, below the pKa of the
conjugated carboxylic acid groups, the LCST was 44.5 1C, an
increase of 5.5 1C over the parent copolymer, whilst at pH 7.4,
above the pKa of the carboxylic acid, a LCST was not observed
up to 100 1C. These results suggest that copolymers displaying
both pH and temperature sensitivity can be accessed with ease
through a simple conjugation procedure.13
11 S. Yamamoto, J. Pietrasik and K. Matyjaszewski, Macromolecules,
2008, 41, 7013–7020.
12 Work by the groups of Brooks/Kizhakkedathu (ref. 2a) and
Schlaad (ref. 2b) indicate that as expected the conjugation of
hydrophobic residues to thermoresponsive polymers results in a
decrease in LCST, although the polymers studied were not based
upon poly-oligo(ethylene glycol) methacrylate.
13 So-called ‘dual responsive’ polymers are a topic of intense research
on account of their potential biomedical applications. For a recent
review see: I. Dimitrov, B. Trebicka, A. H. E. Muller and
¨
A. Dworak, Prog. Polym. Sci., 2007, 32, 1275–1343.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8651–8653 8653