Degradable thermoresponsive polymers which display redox-responsive LCST Behaviour

Article abstract of DOI:10.1039/c1cc16323j

Disulfide linkages were introduced into poly (N-isopropylacrylamide) by the polycondensation of a RAFT-derived, telechelic macromonomer to give degradable yet vinyl-based polymers. These polymers displayed a redox-sensitive lower critical solution tempera

Full text of DOI:10.1039/c1cc16323j

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Degradable thermoresponsive polymers which display redox-responsive
LCST Behaviourw
Daniel J. Phillips and Matthew I. Gibson*
Received 11th October 2011, Accepted 13th November 2011
DOI: 10.1039/c1cc16323j
Disulfide linkages were introduced into poly (N-isopropylacryl-
amide) by the polycondensation of a RAFT-derived, telechelic
macromonomer to give degradable yet vinyl-based polymers.
These polymers displayed a redox-sensitive lower critical
solution temperature (LCST) with the shorter, degraded product
displaying a higher LCST than its non-degraded counterpart.
relevance given the presence of glutathione, a powerful reducing
agent, in vivo. Furthermore, given glutathione is present at
micromolar levels in the systemic circulation, but several
thousand times higher (millimolar) inside cells, this concen-
tration gradient can be exploited to trigger degradation following
cellular internalisation, and has been studied in the field of gene
therapy.6,15 Poly-esters, -amides and -disulfides are generally
accessed by polycondensation or ring opening polymerisation
methods. The low functional group tolerance inherent to these
techniques does however limit the range of possible materials.15–17
Conversely, controlled radical polymerisation techniques such as
reversible addition-fragmentation chain transfer (RAFT) poly-
merisation not only allow the preparation of materials with
exquisite control over molecular weight and polydispersity, but
Macromolecules which display lower critical solution temperature
(LCST) behaviour have attracted significant attention as
‘smart’ materials.1 Heating an aqueous polymer solution
above its LCST initiates an entropically favourable coil-
to-globule transition. This is driven by the expulsion of water
molecules associated with the polymer chain and the formation
of interchain hydrogen bonds. This reversible (or ‘smart’)
behaviour has been exploited, for example, to reversibly
precipitate enzymes,2 to modulate protein catalytic activity3
and to functionalise nanoparticles.4–5 Recently the LCST switch,
which can be considered to modulate polymer hydrophobicity/
lipophilicity, has been investigated as a route to selectively trigger
cellular uptake6–8 and has been demonstrated by the enhanced
uptake of poly (N-isopropylacrylamide), (pNIPAM), in mice
with hyperthermic tumour tissues.9 If in vivo requirements
are to be considered, a macromolecular carrier should have
a molecular weight above the renal filtration limit of approxi-
also enable the incorporation of more advanced functionality.18
A
key challenge therefore is the use of such methodologies to
synthesise biodegradable polymers.
In this contribution, we introduce a new method to generate
reduction-sensitive, degradable polymers from RAFT derived
macromonomers containing a thermoresponsive component.
The polymer is tailored such that following degradation, the
LCST increases dramatically enabling the ‘‘thermosensitivity’’ to
be ‘‘switched off’’, thereby promoting by-product re-solubilisation.
This strategy may also avoid any cytotoxicity associated with
insoluble (above LCST) polymers inside cells/tissue.
mately 40 000 g molꢀ1 10
However, the toxicity associated
.
The thermoresponsive polymer selected in this study was
pNIPAM19 given its biocompatibility and known molecular
weight-dependent LCST behaviour (vide infra).20 Well-defined
pNIPAMs (Mn = 1700, 4400 and 9800 g molꢀ1) were synthe-
sised by RAFT polymerisation (see ESIw) and their cloud
points measured as a function of concentration (Fig. 1).
Polymers with Mn = 4400 and 9800 g molꢀ1 both had cloud
points around 32 1C in the range 1–5 mg mLꢀ1. Interestingly,
when Mn = 1700 g molꢀ1 the cloud point was observed to
increase from 37 to 55 1C as the concentration was decreased
from 5 to 1 mg mLꢀ1. Considering this data, it was decided in
order to obtain the largest possible LCST ‘‘switch’’ following
degradation (vide infra), the molecular weight should shift
with the indefinite accumulation of foreign bodies in tissues
necessitates the inclusion of a degradation mechanism within
the polymer backbone. Polyesters and polyamides can be
degraded by hydrolytic and/or enzymatic processes. However,
these processes are non-specific with the resulting complex
mixture of by-products potentially compromising upon
performance.11 Furthermore, the drop in pH resulting from
carboxylic acid generation can also cause inflammation.12 The
degradation of poly (propylene sulfide), via a non-specific,
oxidative pathway,13 and self-immolating polymers, via a
series of cascade reactions, has also been demonstrated.14 An
alternative strategy is to incorporate reduction-sensitive
disulfide units into the material structure. This is of particular
from above 10 000 g molꢀ1 to below 2000 g molꢀ1
.
The proposed synthesis of a bioreducible, disulfide-containing
pNIPAM from a macromonomer bearing dithioester and
pyridyl disulfide groups at the o- and a-termini respectively
is illustrated in Fig. 2A. This was achieved by exploiting the
inherent end-group control offered by RAFT,21 as described
Department of Chemistry, University of Warwick, Gibbet Hill Road,
Coventry, CV4 7AL, UK. E-mail: m.i.gibson@warwick.ac.uk;
Fax: +44 (0)2476 524112
w Electronic supplementary information (ESI) available including full
experiments and synthetic details. See DOI: 10.1039/c1cc16323j
c
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purified polymer gave Mn = 1300 g molꢀ1 and Mw/Mn = 1.35.
Given the presence of base in the SEC eluent, the formation
of terminal thiols was a possibility. Analysis was therefore
performed in the presence of tributyl phosphine to reduce both
end groups and prevent any potential oxidative- or thiol-pyridyl
disulfide- induced coupling (see ESI for SEC tracesw). 1H NMR
analysis qualitatively indicated the desired dithioester and pyridyl
disulfide end-groups had been installed whilst MALDI-ToF
quantified the presence of both end groups on every chain. This
is shown in Fig. 2B where each peak corresponds to n-NIPAM
repeat units, the mass of both end groups and a lithium/sodium
ion. The molecular weights agree well with the values obtained
by SEC ruling out mass discrimination.
This well-defined macromonomer was subsequently poly-
merised to a disulfide-linked polymer via a polycondensation-
type reaction. 1 equivalent of ethanolamine and 2 equivalents
of triethylamine (TEA) were used to cleave the dithioester at
the o-terminus. The TEA was essential and is hypothesised to
increase the nucleophilicity of the terminal thiol through
deprotonation. To prevent pyridyl disulfide cleavage, the
addition of excess ethanolamine was avoided. Oxygen was
rigorously removed by a minimum of 3 freeze-pump-thaw
cycles to prevent oxidative coupling of terminal thiol groups.
Following 24 h of reaction at 25 1C, SEC analysis revealed a
significant increase in Mw from 1750 to 34 000 g molꢀ1 (Fig. 3).
Given the observed molecular weight was considerably higher
than that expected from oxidative, thiol–thiol coupling reactions
(i.e. dimer formation), the polymerisation was deemed successful.
Thiol-pyridyl disulfide coupling was confirmed as the pre-
dominant mechanism in this case since control experiments,
performed in the presence of oxygen, produced a peak with
Mn = 2600 g molꢀ1, equivalent to oxidative thiol–thiol
coupling.
Fig. 1 Cloud points of pNIPAM as a function of concentration and
molecular weight. Chemical structure of pNIPAM is inset.
by Maynard et al.22 A new RAFT agent, 2-(pyridyldisulfanyl)-
ethyl 4-cyano-4-(phenylcarbonothioylthio)-pentanoate, PECPP,
was used to install these functionalities. PECPP was synthesised
by reaction of hydroxyethyl pyridyl disulfide with 4-cyano-4-
(phenylcarbonothioylthio)pentanoic acid using N,N0-diiso-
propylcarbodiimde as the coupling reagent (for synthetic and
characterisation details, see ESIw). Selective cleavage of the
dithioester to give a o-thiol group can then allow for a poly-
condensation-type reaction of the macromonomer, driven by the
expulsion of pyridine thione.22
NIPAM was polymerised in toluene/methanol (1 : 1) at 70 1C
using PECPP as chain transfer agent. SEC analysis of the
Quantitative removal of the RAFT agent was demonstrated
by use of SEC-coupled photodiode array (see ESIw) and near-
identical molecular weights were obtained when the reaction
was performed at several concentrations (40, 20, 10, 1 mg mLꢀ1).
The presence of disulfides in the backbone was confirmed by
adding the reducing agent tributyl phosphine to the polymer
solution, which resulted in a decrease in molecular weight and a
Fig. 2 (A) Polycondensation using pNIPAM macromonomer: (i)
Ethanolamine (1 molar equiv.); triethylamine (2 molar equiv.); THF;
25 1C; 24 h). (B) MALDI-ToF spectrum of telechelic polymer (inset).
Table shows peak assignments for sodium adducts (indicated in
black). Series of peaks corresponding to lithium adducts (red) are also
shown, and peaks listed in supporting information.w
Fig. 3 SEC analysis demonstrating successful polymer ‘‘condensation’’
procedure using a telechelic macromonomer. Reduction was achieved
using tributyl phosphine.
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glutathione can degrade the polymer, releasing any cargo before
allowing re-solubilisation of the polymer by-products. Future
studies will focus on (i) fine-tuning the transition temperature;
(ii) stimuli-responsive cellular uptake in vitro; (iii) incorporation
of drugs into the polymer side chains via cleavable units.
Equipment used was supported by the Innovative Uses for
Advanced Materials in the Modern World (AM2), with support
from Advantage West Midlands (AWM) and part funded by
the European Regional Development Fund (ERDF). MIG is a
Birmingham Science City Interdisciplinary Research Fellow
funded by the Higher Education Funding Council for England
(HEFCE). Robert C. Deller is thanked for his assistance with
haemolysis assays and Mathew W. Jones for MALDI-ToF.
Notes and references
Fig. 4 Turbidimetry curves showing shift in LCST of pNIPAM
following reduction of backbone disulfide bonds. Red trace is disulfide
linked polymer (Mw = 34 000 g molꢀ1) and blue trace is reduced
polymer (Mw = 1750 g molꢀ1).
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distribution identical to that of the starting material. These
experiments demonstrate that RAFT-derived telechelic macro-
monomers can be used to obtain high molecular weight poly-
meric carriers which specifically degrade in the presence of
reducing agents to well-defined oligomeric by-products.
The primary goal of this work was to use disulfide linkages
as (i) a new route towards selective degradation and (ii) as a
secondary stimulus to shift the LCST of the polymer and
produce soluble by-products. The cloud point of the disulfide-
containing-polymer product was measured before and after
reduction by TCEP (tris-2-carboxyethylphosphine), Fig. 4.
Considering the strong concentration-dependence of this
property, these experiments were conducted at 0.05 mg mLꢀ1
,
close to what might be expected in a biological application.23 The
cloud point shifted from 46 1C to 62 1C following addition of
TCEP and can be directly correlated to the decrease in molecular
weight. Both high and low molecular weight pNIPAMs were
indicated to be biocompatible with red blood cells both above
and below the cloud point up to 3 mg mLꢀ1 (see ESIw).
In conclusion, this manuscript has demonstrated a powerful
new method to incorporate degradable linkages into the back-
bone of RAFT-derived poly (N-isopropylacrylamide), with the
potential for extension to other functional monomers. The
degradation of the polymer promoted a secondary response,
namely an increased LCST following addition of a suitable
reducing agent. This provided an ‘‘off switch’’ allowing the
hydrophilic/hydrophobic transition to be reversed without the
need to lower the solution temperature. This method may find
application for triggered cellular uptake, where intracellular
c
1056 Chem. Commun., 2012, 48, 1054–1056
This journal is The Royal Society of Chemistry 2012