Tweaking the acid-sensitivity of transiently thermoresponsive Polyacrylamides with cyclic acetal repeating units

Article abstract of DOI:10.1007/s11426-019-9705-4

Merging the characteristics of thermoresponsive and stimuli-degradable polymers yields so-called transiently thermoresponsive polymers, which can find application for the design of injectable gels, nanoparticles, etc. within a biomedical context. Among these polymers, only a limited number is reported which shows selective degradation under mild acidic conditions. However, extension of the library of transiently thermoresponsive polymers is desired to broadening the biomaterials toolbox to suit specific needs. Three monomers were developed by modification of 2-hydroxyethylacrylamide (HEAm) via tetrahydropyranylation or -furanylation with 3,4-dihydro-2H-pyran (DHP), 2,3-dihydrofuran (DHF) or 2,3-dihydro-5-methylfuran (MeDHF). The presence of an acetal or ketal bond provided the monomers a pH-dependent hydrolysis behavior ranging from minutes to days. RAFT polymerisation allowed for the construction of homopolymers with temperature responsive behavior and pH-dependent hydrolysis which was strongly influenced by nature of the monomeric repeating units.

Full text of DOI:10.1007/s11426-019-9705-4

SCIENCE CHINA
Chemistry
•
ARTICLES•
https://doi.org/10.1007/s11426-019-9705-4
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tweaking the acid-sensitivity of transiently thermoresponsive
polyacrylamides with cyclic acetal repeating units
*
Simon Van Herck & Bruno G. De Geest
Department of Pharmaceutics, Ghent University, Ghent, Belgium
Received December 10, 2019; accepted February 18, 2020; published online March 13, 2020
Merging the characteristics of thermoresponsive and stimuli-degradable polymers yields so-called transiently thermoresponsive
polymers, which can find application for the design of injectable gels, nanoparticles, etc. within a biomedical context. Among
these polymers, only a limited number is reported which shows selective degradation under mild acidic conditions. However,
extension of the library of transiently thermoresponsive polymers is desired to broadening the biomaterials toolbox to suit
specific needs. Three monomers were developed by modification of 2-hydroxyethylacrylamide (HEAm) via tetra-
hydropyranylation or -furanylation with 3,4-dihydro-2H-pyran (DHP), 2,3-dihydrofuran (DHF) or 2,3-dihydro-5-methylfuran
(MeDHF). The presence of an acetal or ketal bond provided the monomers a pH-dependent hydrolysis behavior ranging from
minutes to days. RAFT polymerisation allowed for the construction of homopolymers with temperature responsive behavior and
pH-dependent hydrolysis which was strongly influenced by nature of the monomeric repeating units.
acetals, polyacrylamides, stimuli-responsive
Citation:
Van Herck S, De Geest BG. Tweaking the acid-sensitivity of transiently thermoresponsive polyacrylamides with cyclic acetal repeating units. Sci
China Chem, 2020, 63, https://doi.org/10.1007/s11426-019-9705-4
1
Introduction
thermoresponsive polymers [10] and stimuli-degradable
polymers [11]. Among the temperature-responsive poly-
mers, polymers that exhibit a coil-to-globule transition
upon change in temperature, those with a lower critical
solution temperature (LCST) have received most attention
[10,12–14]. By far the best known examples are poly(N-
isopropyl acrylamide) (pNIPAm) [15–17] and oligo(ethy-
lene glycol) (meth)acrylate) [18] polymers. Block copoly-
mer systems based on these polymers have the ability to
self-assemble into nanostructures upon heating in aqueous
environment, thus avoiding the use of organic solvents to
form drug delivery vehicles. The main limitation of the
above-mentioned polymers is that they lack the property to
disassemble, hindering both the release of active compound
and the clearance from the body. The hindrance in drug
release has given rise to the second type, the stimuli-de-
gradable polymers. Incorporation of functional groups that
are sensitive to localized conditions like pH, oxidation/re-
The evolution in materials chemistry has led to broad range
of materials that hold promise for applications in a drug
delivery [1]. Many examples exist of protein therapeutics
which are PEGylated to reduce opsonisation and increase
circulation time [2,3]. Moreover, polymers can be utilized to
encapsulate drugs into nanocarriers [4,5]. Encapsulation of
highly potent compounds circumvents the poor aqueous
solubility often seen with these compounds and leads to a
more advantageous safety profile due to improved pharma-
cokinetics and pharmacodynamics [6–9]. Despite the vast
amount of research in this field, successful clinical transla-
tion is rather limited [6].
For drug delivery applications, two classes of polymers
have received a formidable amount of attention, being
*Corresponding author (email: br.degeest@ugent.be)
©
Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
chem.scichina.com link.springer.com

2
Van Herck et al. Sci China Chem
duction or specific enzymes can provide on-demand drug
delivery [19–22].
2.2 Instrumentation
1
13
All H- and C-NMR spectra were recorded on a Bruker
00 MHz or 400 MHz FT NMR spectrometer (Germany).
A combination of the two responsive properties is pro-
vided by so-called transiently thermoresponsive polymers—
i.e., polymers that change their conformation in response to
temperature, but loose this property as consequence to an
alteration in the polymer side chain or backbone induced by a
physiologically relevant stimulus rendering them fully hy-
drophilic [10,23]. This concept allows to start with a soluble
polymer that upon temperature increase will self-assemble
into a micellar structure able to incorporate a hydrophobic
compound and that after endocytosis will become fully so-
luble irrespective of temperature due to hydrolysis of in the
side chains induced by the acidic pH in the endo/lysosomal
vesicles.
To allow for endo/lysosomal acidic-pH induced hydro-
lysis, acetal chemistry has been explored [24]. A large body
of research has been devoted to polyacetal/ketal synthesis by
step-growth polymerization [25–31], whereas only a limited
number of transiently thermoresponsive polymers systems
with an acid-dependent degradation have been reported
3
Chemical shifts (δ) are provided in ppm relative to TMS.
Samples were prepared in given deuterated solvents and their
signals referenced to residual non-deuterated signals of the
solvent.
ESI-MS was performed on a Waters LCT Premier XETM
time of flight (TOF) mass spectrometer (USA) equipped with
a standard electrospray ionization and modular LockSpray
TM interface. The purity of the products was assessed by
high-performance liquid chromatography (HPLC) and pho-
todiode array (PDA) detection (190–400 nm) using a reverse
phase column (Phenomenex Luna 3 µm C18 (2), 100 Å,
2
00 mm) with a linear gradient of 10%–100% B over 9 min,
where A is 0.1% formic acid in H O and B is 0.1% formic
2
acid in CH CN at a flow rate of 0.4 mL/min.
3
HPLC measurement was performed using a system with an
isocratic solvent pump (L-7100, Merck, Hitachi LaChrom,
Japan), an autosampler (L-7200, Merck, Hitachi LaChrom)
with a loop of 100 µL, a guard column (RP 18e) followed by
a reversed phase C18 column (LiChroCart® 125-4, Li-
Chrospher® 100 RP (5 μm)) and a UV-detector (L-7400,
Merck, Hitachi LaChrom).
Molecular weight distribution analysis was performed
by size exclusion chromatography (SEC) measurements
on a Shimadzu 20A system in dimethylacetaminde
[10,32]. These either consist of a degradable monomer co-
polymerised with a non-degradable thermoresponsive
monomer or do not possess the desirable properties in a
biologically relevant window [32,33].
Hence, extending the limited library of currently existing
transiently thermoresponsive polymers is needed to obtain
polymers with more optimal properties as well as broad-
ening the biomaterials toolbox to suit specific needs. Here
we present a new set of transiently thermoresponsive
polymers that exhibit LCST behavior and degrade into fully
water soluble polymers in response to an acidic stimulus.
We build on previous work published by our group on a 3,4-
dihydro-2H-pyran (DHP) modified acrylamide by extend-
ing the modifications with furan acetal and ketal derivatives
(
DMAc) as solvent containing 50 mM LiBr. The system
was equipped with a 20A ISO-pump and a 20A refractive
index detector (RID). Measurements were recorded at
5
2
0 °C with a flow rate of 0.700 mL/min. Calibration of the
PL 5 μm Mixed-D columns was done with poly(methyl
methacrylate) (PMMA) standards obtained from PSS
Polymer Standards Service GmbH (Mainz, Germany).
Samples were run with toluene as an internal standard.
Dynamic light scattering (DLS) was performed on a Ze-
tasizer Nano S (Malvern Instruments Ltd., Malvern, UK)
equipped with a HeNe laser (λ=633 nm) and detection at
scattering angle of 173°. Cumulants analysis of the data gave
the z-average and polydispersity index and data fitting by
CONTIN the particle size distribution.
[
34]. A comparative study is performed to investigate the
influence of hydrophobic modification on the relevant
properties like the phase-transition temperature (for the
sake of simplicity denoted as cloud-point temperature, Tcp),
hydrolysis rate and self-assembly behavior of polymers
made thereof.
2
Experimental
2.3 Methods
2
.1 Materials
Synthesis of N-(2-((tetrahydro-2H-pyran-2-yl)oxy])ethyl)-
acrylamide (THPEAm). Camphorsulfonic acid (1.630 g,
7.02 mmol) and N-(2-hydroxyethyl)acrylamide (7.5 mL,
70.2 mmol) were dissolved in 100 mL dry dichloromethane
(DCM). To this mixture 3,4-dihydro-2H-pyran (7.68 mL,
70.2 mmol) was added dropwise at 0 °C. The reaction mix-
All chemicals and solvents were obtained from commercial
sources and used as perceived unless otherwise noted. 2.2′-
azobis(2-methylpropionitrile) (AIBN) as initiator was pro-
vided by WAKO Chemicals and purified by recrystallization
from diethyl ether prior to use. The RAFT CTA 2-(bu-
tylthiocarbonothioylthio)propanoic acid (PABTC) was syn-
thesized according to literature [35,36].
ture was stirred at RT under N . After 3 h the reaction is
2
quenched by addition of trieyhylamine (TEA). The mixture

Van Herck et al. Sci China Chem
3
was concentrated in vacuo, dissolved in ethyl acetate and
filtered. The filtrate was concentrated and purified by col-
umn chromatography (DCM/EtOAc–80:20+1% TEA) to
2.4 Determination of hydrolysis rate of acetal bearing
monomers (THPEAm and THFEAm)
1
Monomer solutions were prepared at 0.5 mg/mL in 100 mM
acetate buffer pH 5 and 100 mM phosphate buffer pH 7.4 in
triplicate. A trace amount of hydroquinone monomethyl
ether (200 ppm) was added to avoid polymerisation. The
solutions were stirred at 37 °C. At regular time points sam-
ples were taken by diluting 20 µL of the hydrolysis solution
into 180 µL 100 mM phosphate buffer pH 7.4 and stored at
give 11.9 g (85% yield) of a clear viscous oil. H NMR
(
300 MHz, DMSO-d ) δ ppm: 8.16 (br.s, 1H), 6.25 (dd,
6
J=17.1, 10.0 Hz, 1H), 6.07 (dd, J=17.1, 2.4 Hz, 1H), 5.57
dd, J=10.0, 2.4 Hz, 1H), 4.56 (t, J=3.7 Hz, 1H), 3.74 (tt,
J=8.0, 3.2 Hz, 1H), 3.64 (dt, J=9.7, 6.0 Hz, 1H), 3.45–3.37
(
(
m, 2H), 3.33–3.21 (m, 2H), 1.82–1.67 (m, 1H), 1.67–1.57
13
(m, 1H), 1.55–1.35 (m, 4H). C NMR (75 MHz, DMSO-d )
6
−
18 °C. Prior to injection the samples were diluted 5 times
δ ppm: 164.67, 131.73, 125.03, 98.06, 65.41, 61.40, 38.72,
with eluent. Analysis was done by HPLC using water/acet-
onitrile 60:40 as eluent, with the flow rate set at 0.2 mL/min
and detection at 207 nm. Assessment of the hydrolysis rate
was done taking the ratio of molar concentrations of the
compounds calculated from calibration curves for THPEAm
and HEAm (Figure S8).
3
0.20, 25.00, 19.15. ESI-MS calcd for C H NO , m/z
10 17 3
+
=
222.1101, found 222.1111 [M+Na] .
Synthesis of N-(2-((tetrahydro-2H-furan-2-yl)oxy)ethyl)
acrylamide (THFEAm). Camphorsulfonic acid (921.8 mg,
-
3
3
.968 mmol) and N-(2-hydroxyethyl)acrylamide (4.568 g,
9.68 mmol) were dissolved in 80 mL dry DCM. To this
[
HEAm]
mixture 2,3-dihydrofuran (3.0 mL, 39.68 mmol) was added
dropwise at 0 °C. The reaction mixture was stirred at RT
%
hydrolysis=
× 100%
(
[HEAm] +[HEAmTHP])
under N . After 3 h the reaction is quenched by addition of
2
TEA. The mixture was concentrated in vacuo and purified by
2.5 Determination of hydrolysis rate of MeTHFEAm
column chromatography (Hex/EtOAc–50:50+1% TEA) to
1
Monomer solution was prepared at 0.5 mg/mL in 20 mM
acetate buffer (pH 5), 20 mM phosphate buffer (pH 7.4) and
in 20 mM carbonate buffer (pH 9) in triplicate. A trace
amount of hydroquinone monomethyl ether (200 ppm) was
added to avoid polymerisation. The solutions were stirred at
give 5.029 g (68% yield) of a clear oil. H NMR (400 MHz,
DMSO-d ) δ ppm: 8.11 (s, 1H), 6.24 (dd, J=17.1, 10.1 Hz,
6
1
1
5
1
H), 6.07 (dd, J=17.1, 2.3 Hz, 1H), 5.57 (dd, J=10.1, 2.3 Hz,
H), 5.11–5.05 (m, 1H), 3.80–3.70 (m, 2H), 3.56 (dt, J=9.9,
.9 Hz, 1H), 3.41–3.35 (m, 1H), 3.32–3.18 (m, 2H), 1.91–
3
7 °C. At regular time points, 4 µL sample was collected and
13
.81 (m, 2H), 1.80–1.68 (m, 2H). C NMR (101 MHz,
diluted to 200 µL with mobile phase. All samples were im-
mediately stored in the freezer before further quantification
by HPLC. HPLC analysis was done using carbonate buffer
DMSO) δ ppm: 164.64, 131.73, 125.02, 103.22, 66.17,
5.11, 38.75, 31.81, 23.03. ESI-MS calcd for C H NO ,
6
9
15
3
+
m/z=208.0944, found 208.0943 [M+Na] .
(pH 9)/acetonitrile–70:30 as mobile phase, flow rate at
Synthesis of N-(2-((2-methyltetrahydrofuran-2-yl)oxy)
ethyl)acrylamide (MeTHFEAm). To N-(2-hydroxyethyl)ac-
rylamide (8.55 g, 71.3 mmol) dissolved in 300 mL anhy-
drous DCM were consecutively added molecular sieves
followed by 2,3-dihydro-5-methylfuran (5.0 g, 59.4 mmol)
and the mixture was cooled on ice. Camphorsulfonic acid
0
.2 mL/min and detection at 207 nm. Assessment of the
hydrolysis rate was done taking the ratio of the AUC for
hydrolysis product (HEAm) over the total AUC of hydro-
lysis product+starting product, according to following
equation:
AUCHEAm
AUCMeTHFEAm +AUCHEAm)
(
138 mg, 0.59 mmol) was added and the reaction mixture
%
hydrolysis=
× 100%
(
was stirred 30 min on ice followed by 3 h at room tem-
perature. Next, the reaction was quenched with TEA
RAFT homopolymerisation of THPEAm. pTHPEAm and
42
(
8.3 mL, 59.4 mmol), filtered, concentrated under reduced
pTHPEAm66 were synthesised following the same protocol
with only altering the amount of PABTC and AIBN. As an
example, the protocol for pTHPEAm42 is given here. A
Schlenk tube was loaded with THPEAm (600 mg, 3 mmol),
pressure and purified by column chromatography (hexane/
EtOAc–20:80+1% TEA) to give 6.97 g (58%) of an yel-
lowish oil. H NMR (400 MHz, DMSO-d ) δ ppm: 8.09 (br.
1
6
s, 1H), 6.23 (dd, J=17.1, 10.1 Hz, 1H), 6.07 (dd, J=17.1,
2
-(butylthiocarbonothioylthio)propanoic acid (PABTC)
2
2
2
.3 Hz, 1H), 5.56 (dd, J=10.1, 2.3 Hz, 1H), 3.82–3.66 (m,
H), 3.47–3.33 (m, 2H), 3.26–3.18 (m, 2H), 2.01–1.87 (m,
H), 1.86–1.74 (m, 1H), 1.72–1.61 (m, 1H), 1.33 (s, 3H).
(
14.35 mg, 0.06 mmol) and AIBN (1.97 mg, 0.012 mmol)
and dissolved in anhydrous DMF (2 M). The mixture was
degassed by five freeze-vacuum-thaw cycles and immersed
in a pre-heated oil bath at 80 °C under vigorous stirring.
After 1 h the reaction was quenched by cooling and exposure
to air. Samples were taken at regular time point and analysed
1
3
C NMR (101 MHz, DMSO-d ) δ ppm: 164.61, 131.78,
6
1
24.93, 106.99, 59.25, 39.19, 37.38, 24.02, 21.95. ESI-MS
calcd for C H NO , m/z=222.1101, found 222.1122 [M
+
1
0
17
3
1
+
by H NMR to determine monomer conversion. The result-
Na] .

4
Van Herck et al. Sci China Chem
ing polymer was isolated by repeated precipitations in ice-
cold diethyl ether with acetone for re-dissolving the polymer.
The precipitated polymer was dried under vacuum to give
2.6 Cloud point determination of homopolymers
Polymer dispersions were made at 5 mg/mL in PBS or saline
NH OH solution (7.5 mM ammonia+150 mM NaCl) and
4
3
00 mg yellow powder. Polydispersity of purified polymer
1
cooled. The cooled solutions were filtered through a
was analysed by size exclusion chromatography (SEC). H
0
.450 µm filter before measurement to remove dust. Scat-
NMR (300 MHz, DMSO-d ) δ ppm 4.63 (s, 1H), 3.90 (s,
6
tering intensity and size were measured over a temperature
trend with interval of 1 °C by DLS. Three repeated mea-
surements were done at each temperature except for
pMeTHFEAm polymers due to sensitivity to hydrolysis.
1
9
H), 3.80 (s, 1H), 3.68–3.32 (m, 4H), 1.65 (d, J=54.7 Hz,
H).
Conditions for pTHPEAm . THPEAm (600 mg, 3 mmol),
66
2
0
-propanoic acid butyl trithiocarbonate (7.18 mg,
.03 mmol) and AIBN (0.988 mg, 0.006 mmol).
RAFT homopolymerisation of THFEAm. pTHFEAm and
2.7 Determination of hydrolysis rate of THPEAm
homopolymers
4
7
pTHFEAm91 were synthesised following the same protocol
with only altering the amount of PABTC and AIBN. As an
example, the protocol for pTHFEAm47 is given here. A
Schlenk tube was loaded with THFEAm (1 g, 5.4 mmol), 2-
propanoic acid butyl trithiocarbonate (25.74 mg,
pTHPEAm hydrolysis mixtures were prepared by dissolving
the polymer in cold buffer. The experiment is started by
heating to 37 °C while stirring. A 50 mM acetate buffer pH 5
+
1 M NaSCN and 50 mM phosphate buffer pH 7.4+1 M
0
.108 mmol) and AIBN (3.547 mg, 0.0216 mmol) and dis-
NaSCN were used as testing conditions. At regular time
points a 4 mL sample was taken and dialysed against 10 mM
solved in anhydrous DMF (2 M). The mixture was degassed
with five freeze-vacuum-thaw cycles and immersed in a pre-
heated oil bath at 80 °C under vigorous stirring. After 1 h, the
reaction was quenched by cooling and exposure to air.
NH OH solution. All samples were dialysed 4 d during
4
which the medium was frequently refreshed, followed by
freeze drying. The freeze-dried samples were dissolved in
1
Samples were taken at regular time point and analysed by H
1
deuterated methanol for H NMR analysis, afterwards me-
NMR to determine monomer conversion. The resulting
polymer was isolated by repeated precipitations in ice-cold
diethyl ether with acetone for re-dissolving the polymer. The
precipitated polymer was dried under vacuum to give
thanol was evaporated, and 5 mg/mL sample solutions were
made in 50 mM phosphate buffer pH 7.4 for turbidimetry
measurement. Measurements were done by 3 repeated
heating-cooling cycles and the average was used to plot the
graphs.
5
84 mg yellow powder. Polydispersity of purified polymer
1
was analysed by size exclusion chromatography (SEC). H
NMR (300 MHz, DMSO-d ) δ ppm 7.37 (s, 1H), 5.09 (d, J=
6
5
2
.0 Hz, 1H), 3.76 (t, J=6.3 Hz, 2H), 3.51 (s, 2H), 3.18 (s,
H), 2.20–1.04 (m, 7H).
2.8 Determination of hydrolysis rate of pTHFEAm and
pMeTHFEAm
RAFT homopolymerisation of MeTHFEAm. pMeTH-
Hydrolysis of pTHFEAm and pMeTHFEAm polymers was
monitored by H NMR. Polymers were dispersed at
FEAm and pMeTHFEAm were synthesised following the
43
46
1
same protocol with only altering the amount of PABTC and
AIBN. As an example, the protocol for pMeTHFEAm43 is
given here. A Schlenk tube was loaded with MeTHFEAm
741 mg, 4 mmol), 2-propanoic acid butyl trithiocarbonate
19.1 mg, 0.08 mmol) and AIBN (2.63 mg, 16 µmol) and
1
0 mg/mL in deuterated phosphate buffers with pH 5, 7 or 9
and incubated at room temperature. At regular time points
measurements were collected.
(
(
dissolved in anhydrous DMF (2 M) containing 10 V% pyr-
idine. The mixture was degassed with five freeze-vacuum-
thaw cycles and immersed in a pre-heated oil bath at 80 °C
under vigorous stirring. After 75 min the reaction was
quenched by cooling and exposure to air. Samples were ta-
3
Results and discussion
3
.1 Monomer synthesis
Previously, we reported on engineering the water-soluble
acrylamide N-(2-hydroxyethyl)acrylamide (HEAm), a com-
mercially available hydrophilic monomer, with an acid-
sensitive hydrophobic moiety through an acetal using DHP,
yielding THPEAm. In our endeavours to design transiently
thermoresponsive polymers, the use of acrylamides instead
of (meth)acrylates, takes advantage of the presence of the
amide moiety which provides the repeating units with a more
hydrophilic character through hydrogen bonding with water
molecules below the phase transition temperature of the re-
1
ken at regular time point and analysed by H NMR to de-
termine monomer conversion. The resulting polymer was
isolated by repeated precipitations in a mixture of ice-cold
diethyl ether/hexane with acetone for re-dissolving the
polymer. The precipitated polymer was dried under vacuum
to give a yellow powder. Polydispersity of purified polymer
1
was analysed by size exclusion chromatography (SEC). H
NMR (400 MHz, DMSO-d ) δ ppm 7.37 (s, 1H), 3.87–3.65
6
(m, 2H), 3.41 (s, 2H), 3.14 (s, 2H), 2.11–1.17 (m, 10H).

Van Herck et al. Sci China Chem
5
sulting polymers. However, dehydration of the acrylamide
3.2 Acid catalysed monomer degradation
side chains on its turn should provide a driving force for coil-
to-globule transition above a critical Tcp [14,37]. Here we
extended the monomer toolbox by reacting HEAm with re-
spectively 2,3-dihydrofuran (DHF) and 2,3-dihydro-5-me-
thylfuran (MeDHF), yielding N-(2-((tetrahydro-2H-furan-2-
yl)oxy)ethyl)-acrylamide (THFEAm) and N-(2-((2-methyl-
tetrahydrofuran-2-yl)oxy)ethyl)acrylamide (MeTHFEAm),
respectively (Scheme 1). DHP is a well-known compound
from protection group chemistry [38]. It is used as a base-
stable protective moiety for hydroxyl groups obtained
through tetrahydropyranylation and can be cleaved under
acidic conditions due to hydrolysis of the acetal bond. In this
context we expanded this approach with two lesser known
furan based compounds, which allowed us to investigate the
influence of a 5 or 6 member ring (THP vs. THF) and an
acetal or ketal bond (THP/THF vs. MeTHF).
Acid catalysed hydrolysis of all three monomers was con-
firmed by H NMR analysis (Figure 1), showing a shift of the
1
acetal proton (b) to a lactol proton (c) for THPEAm and
THFEAm and the disappearance of the furan ring (f) for
MeTHFEAm. The pH-dependent hydrolysis rate of the dif-
ferent functionalities was evaluated by HPLC analysis in
response to aqueous buffers at pH 5, 7.4 and 9 representing
lysosomal, physiological and basic pH conditions, respec-
All monomers could be obtained through an acid-catalysed
acetalization reaction between the hydroxyl group of HEAm
and the enol ether in the pyran or furan ring. Reactions were
conducted at low temperature using equimolar amounts of
both reagents and a catalytic amount of camphorsulfonic
acid (CSA). The reaction yields strongly differed based on
the nature of the chemical bond, with the lowest yield ob-
tained for the more acid-sensitive ketal linkage (MeTH-
FEAm, 58%). NMR spectra of the purified monomers are
provided in Figures S1–S6 (Supporting Information online).
In an attempt to further optimise monomer synthesis an al-
ternative reaction was tested using an organocatalyzed
acetalization described by Kotke et al. [39,40]. Their
“
Schreiner’s Catalyst” is an electron-deficient thiourea able
to catalyse the acetal formation under non-acidic conditions,
thereby avoiding the equilibrium status of the acetalization
which lowers product yield. However, no advantage was
observed compared to the acid-catalysed reaction, at least not
in our hands. Reactions done in dilution media showed ex-
tremely low kinetics and the use of a large excess of vinyl
ether yielded side reactions. This route was therefore not
investigated further.
1
Figure 1 H NMR (D O or DMSO-d ) of modified acrylamide mono-
2
6
mers: THPEAm (A), THFEAm (B) and MeTHFEAm (C) before and after
hydrolysis in acidic conditions. Most relevant proton peaks for identifica-
tion of the hydrolysis products are marked in red (color online).
Scheme 1 Monomer synthesis and RAFT polymerization of THPEAm,
THFEAm and MeTHFEAm.

6
Van Herck et al. Sci China Chem
tively (Figure 2). Although THPEAm and THFEAm bear
great similarity in structure, a clear difference was observed
in degradation rate under acidic conditions (i.e., pH 5 buffer).
THFEAm has a half-life of approximately 5 h, while this was
hydrolysis was rather fast with a half-life around 9 h, pro-
viding proof for the instability of the ketal group of
MeTHFEAm in aqueous environment. This is in sharp
contrast with the acetal containing monomers, THPEAm and
THFEAm, that show only limited hydrolysis at pH 7 after
several days. The much faster hydrolysis rate is known for
ketal bonds and is additionally driven by the equilibrium
between cyclic (Figure 1(c) shift f) or linear state (Figure 1
(c) shift d/g) of the hydrolysis product, being predominantly
the linear 5-hydroxy-2-pentanone [41].
1
9 h for THPEAm. Both the difference in hydrophobicity
and/or ring strain could explain this behavior. Figure 1(b)
shows that the higher ring strain of the 5-membered lactol
ring formed during hydrolysis of THFEAm causes an in-
creased amount of ring opening and formation of the corre-
sponding aldehyde, 4-hydroxybutanal (shift d). This on its
turn could shift the equilibrium towards hydrolysis as there is
a decreased amount of direct hydrolysis product, i.e., the
lactol. Hydrolysis of THPEAm (Figure 1(a)) only yields up
to 2.5% of the 5-hydroxypental, while up to 15% of 4-hy-
droxybutanal is formed upon THFEAm (Figure 1(b)) hy-
drolysis. The switch from an acetal to a ketal bond for
MeTHFEAm results in a dramatic increase in hydrolysis
rate. Full hydrolysis of MeTHFEAm was immediate in pH 5
buffer and occurred within the hour at pH 7. Even at pH 9
3
.3 Polymerization and characterization
Homopolymers of the three acrylamide monomers were
synthesised by reversible addition-fragmentation chain
transfer (RAFT) polymerization using PABTC as chain
transfer agent (CTA), aiming at a degree of polymerisation
(DP) of 50 and 100 (Scheme 1 and Table 1). Due to the
extreme sensitivity of MeTHFEAm to degradation of the
MeTHF moiety, pyridine was added as a base to protect the
monomer against hydrolysis. Pyridine was selected over
other organic bases such as TEA or DBU as the latter two
either stopped polymerisation at low conversion or inhibited
1
polymerisation all together. As an example, the H NMR
spectra of the purified polymers are shown in Figures S8–
S10. Monomer conversion was monitored over time during
polymerisation to analyse the effect of functionalisation on
the polymerisation kinetics (Figure 3 and Figures S11–S13).
From the kinetics of monomer consumption, it is clear that
polymerisation of THFEAm is faster compared to THPEAm
and MeTHFEAm. Although initial polymerisation rates for
THPEAm and THFEAm are fairly similar, at high conver-
sion, the polymerisation rate of THPEAm is markedly
slower. This can possibly be linked to steric effect or to
viscosity related effects, with higher viscosity for the bulkier
THPEAm. Structural effects are even more pronounced at
monomer:CTA ratios of 100:1. With both THPEAm and
MeTHFEAm showing a delayed initiation and a much
slower polymerisation rate compared to THFEAm.
3
.4 Evaluation thermoresponsive properties
The thermoresponsive properties of the homopolymers were
measured by DLS at polymer concentrations of 5 mg/mL in
phosphate buffered saline (PBS; pH 7.4, 0.15 M NaCl) or in
a saline ammonia buffer (0.1% NH OH+0.15 M NaCl)
4
(Figure 4, Table 1 and Figure S14). All polymers showed a
temperature-induced phase transition below physiological
temperature. Above this Tcp polymers, formed large in-
soluble aggregates in aqueous medium due to transition into
fully hydrophobic polymer chains. As seen from the Tcp
values in Table 1 as well as in Figure 4, the modification had
Figure 2 pH-Dependent hydrolysis profiles of modified acrylamide
monomers analysed by HPLC (a) and homopolymers analysed by H NMR
1
(b). (c) Relative hydrolysis rates of monomers and homopolymers given as
the half-life as a function of pH (color online).
a strong effect on the T , with values well below room
cp

Van Herck et al. Sci China Chem
7
Figure 3 Kinetics plots of polymerisation of THPEAm (a1), THFEAm (a2) and MeTHFEAm (a3) via RAFT polymerisation. (b) Molar mass distribution
profiles of purified homopolymers analysed by size exclusion chromatography in DMAc (color online).
Table 1 Summary of polymerization conditions and characterization of synthesized homopolymers
sec
theor
a)
M (eq. to
CTA)
Temp.
(°C)
Conv. DP from
Mn
Mn
(kD)
b)
Polymer
CTA
Solvent
Time (h)
a)
b)
ᴆ
Tcp (°C)
(%)
84
66
94
91
84
46
conv.
(kD)
c
pTHPEAm42
pTHPEAm66
PABTC
PABTC
PABTC
PABTC
PABTC
50
100
50
DMF
DMF
80
80
80
80
80
80
1 h
42
7.4
8.6
1.12
1.12
1.17
1.14
1.19
1.18
8
c
1 h
66
12.1
6.1
13.4
8.9
6
c
pTHFEAm50
DMF
75 min
75 min
75 min
75 min
47
32
c
pTHFEAm75
100
50
DMF
91
12.2
6.4
17.1
8.6
27
d)
pMeTHFEAm42
pMeTHFEAm46
DMF+10 V% pyridine
DMF+10 V% pyridine
42
29
d
PABTC
100
46
8.0
9.4
29
1
a) Calculated based on H NMR spectroscopy data. b) Determined by SEC in DMAc using PMMA for calibration. c) Determined by DLS at 5 mg/mL in
PBS. d) Determined by DLS at 5 mg/mL in 10 mM NH OH solution+150 mM NaCl.
4
temperature for pTHPEAm and above room temperature for
pTHFEAm and pMeTHFEAm. Although this was never
directly investigated, different experimental observations
suggest the larger 6-membered pyran ring to be much more
monomer. This was not the case for pTHPEAm which ex-
hibited a half-life of 48 h, compared to 19 h for the THPEAm
monomer (Figure 1(C)). The decrease in hydrolysis rate at
pH 5 observed for pTHPEAm can be attributed to the limited
aqueous solubility of the polymer with decreased accessi-
bility of water molecules to the collapsed polymer coils
hydrophobic, resulting in a lower T . Within the same
cp
modification, some influence of the chain length on the Tcp
was observed albeit with a minimal shift. In general, an in-
crease in degree in polymerization resulted in a decrease in
Tcp due to a more hydrophobic nature of the polymer. This is
in line with earlier observations by us on the influence on
hydrophobic block length on the Tcp for thermoresponsive
block copolymers [7,34,42].
above its T . The transiently thermoresponsive behavior—i.e.,
cp
a gradual shift in Tcp upon hydrolysis of the pending THP
moieties—was investigated by turbidimetry on a colloidal
suspension of pTHPEAm at 37 °C (Figure 5 and Figure S15).
A clear shift in T was found which proves that pTHPEAm
cp
loses its hydrophobic properties to become fully soluble,
irrespective of temperature. A detailed investigation of the
transiently thermoresponsive behavior was not performed for
the other polymers due to practical considerations, with
pMeTHFEAm hydrolysis to fast and pTHFEAm having a
The pH-dependent hydrolysis was evaluated for the
1
homopolymers by H NMR and revealed a similar trend as
observed for the corresponding monomers, showing im-
mediate degradation of the ketal side chain in pMeTHFEAm,
both at pH 5 and pH 7 (Figure 1(B)). The degradation half-
life for pTHFEAm was in the same range as the THFEAm
too high T at the start. Too high in this context indicates too
cp
close to physiological temperature.

8
Van Herck et al. Sci China Chem
Figure 5 Transmittance profiles of pTHPEAm42 dispersions in function
of temperature at different time points during hydrolysis at pH 5 (a1) and
pH 7.4 (a2) analysed by turbidimetry (color online).
Acknowledgements
This work was supported by Ghent University
through the BOF-GOA grant scheme.
Conflict of interest The authors declare that they have no conflict of
interest.
Supporting information The supporting information is available online at
http://chem.scichina.com and http://link.springer.com/journal/11426. The
supporting materials are published as submitted, without typesetting or
editing. The responsibility for scientific accuracy and content remains en-
tirely with the authors.
Figure 4 Mean particle diameter (red) and scattering intensity (blue)
profiles in function of temperature of homopolymers with targeted DP of
1
2
3
4
Ringsdorf H. J Polym Sci C Polym Symp, 2007, 51: 135–153
Harris JM, Chess RB. Nat Rev Drug Discov, 2003, 2: 214–221
Owensiii D, Peppas N. Int J Pharm, 2006, 307: 93–102
5
0 analysed by DLS at 5 mg/mL in PBS or saline NH OH buffer (color
4
online).
Davis ME, Chen ZG, Shin DM. Nat Rev Drug Discov, 2008, 7: 771–
4
Conclusions
782
5
6
Moses MA, Brem H, Langer R. Cancer Cell, 2003, 4: 337–341
Louage B, De Wever O, Hennink WE, De Geest BG. J Control Re-
lease, 2017, 253: 137–152
Louage B, Zhang Q, Vanparijs N, Voorhaar L, Vande Casteele S, Shi
Y, Hennink WE, Van Bocxlaer J, Hoogenboom R, De Geest BG.
Biomacromolecules, 2015, 16: 336–350
Kasmi S, Louage B, Nuhn L, Van Driessche A, Van Deun J, Karalic I,
Risseeuw M, Van Calenbergh S, Hoogenboom R, De Rycke R, De
Wever O, Hennink WE, De Geest BG. Biomacromolecules, 2016, 17:
Summarizing, cyclic acetal and ketal-functionalized acryla-
mide monomers were obtained through the modification of
the hydrophilic 2-hydroxyethyl acrylamide (HEAm). All
monomers could be polymerised by RAFT yielding well
defined homopolymers and block copolymers with transient
thermoresponsive behavior. Monomers and polymers all
showed a pH-dependent hydrolysis due to the presence of the
acetal/ketal bond, with accelerated hydrolysis at endosomal
pH. Phase transition temperatures strongly varied with
modification and chain length of the hydrophobic segment.
The more hydrophobic THPEAm polymers resulted in Tcp
values well below room temperature while these were much
higher for THFEAm and MeTHFEAm polymers. We an-
ticipate this work provides a rational base for the design of
supramolecular materials containing transiently responsive
polymeric building blocks.
7
8
119–127
9
0
Shi Y, van der Meel R, Theek B, Oude Blenke E, Pieters EHE, Fens
MHAM, Ehling J, Schiffelers RM, Storm G, van Nostrum CF, Lam-
mers T, Hennink WE. ACS Nano, 2015, 9: 3740–3752
Vanparijs N, Nuhn L, De Geest BG. Chem Soc Rev, 2017, 46: 1193–
1239
1
11 Tong R, Tang L, Ma L, Tu C, Baumgartner R, Cheng J. Chem Soc Rev,
014, 43: 6982–7012
Roy D, Brooks WLA, Sumerlin BS. Chem Soc Rev, 2013, 42: 7214–
243
2
1
2
7
13 Stuart MAC, Huck WTS, Genzer J, Müller M, Ober C, Stamm M,
Sukhorukov GB, Szleifer I, Tsukruk VV, Urban M, Winnik F, Zau-

Van Herck et al. Sci China Chem
9
scher S, Luzinov I, Minko S. Nat Mater, 2010, 9: 101–113
Gil E, Hudson S. Prog Polym Sci, 2004, 29: 1173–1222
Zhang Z, Maji S, da Fonseca Antunes AB, De Rycke R, Hoogenboom
R, De Geest BG. Angew Chem Int Ed, 2016, 55: 7086–7090
Schild HG. Prog Polym Sci, 1992, 17: 163–249
29 Lingier S, Nevejans S, Espeel P, De Wildeman S, Du Prez FE.
Polymer, 2016, 103: 98–103
30 Delplace V, Nicolas J. Nat Chem, 2015, 7: 771–784
31 Wang Y, Huang D, Wang X, Yang F, Shen H, Wu D. Biomater Sci,
2019, 7: 3238–3248
32 Vanparijs N, De Coen R, Laplace D, Louage B, Maji S, Lybaert L,
Hoogenboom R, De Geest BG. Chem Commun, 2015, 51: 13972–
13975
33 Zou Y, Brooks DE, Kizhakkedathu JN. Macromolecules, 2008, 41:
5393–5405
34 Van Herck S, Van Hoecke L, Louage B, Lybaert L, De Coen R, Kasmi
S, Esser-Kahn AP, David SA, Nuhn L, Schepens B, Saelens X, De
Geest BG. Bioconjugate Chem, 2018, 29: 748–760
35 Ferguson CJ, Hughes RJ, Nguyen D, Pham BTT, Gilbert RG, Serelis
AK, Such CH, Hawkett BS. Macromolecules, 2005, 38: 2191–2204
36 Van Herck S, Hassannia B, Louage B, Pita Compostizo R, De Coen R,
Vanden Berghe W, Vanden Berghe T, De Geest BG. Eur Polym J,
2019, 110: 313–318
1
1
4
5
1
1
6
7
Yin L, He C, Huang C, Zhu W, Wang X, Xu Y, Qian X. Chem
Commun, 2012, 48: 4486
1
1
2
8
9
0
Lutz JF. J Polym Sci A Polym Chem, 2008, 46: 3459–3470
Mura S, Nicolas J, Couvreur P. Nat Mater, 2013, 12: 991–1003
Tang L, Zheng Y, Melo MB, Mabardi L, Castaño AP, Xie YQ, Li N,
Kudchodkar SB, Wong HC, Jeng EK, Maus MV, Irvine DJ. Nat
Biotechnol, 2018, 36: 707–716
Liu B, Thayumanavan S. J Am Chem Soc, 2017, 139: 2306–2317
Zheng L, Zhang X, Wang Y, Liu F, Peng J, Zhao X, Yang H, Ma L,
Wang B, Chang C, Wei H. Biomacromolecules, 2018, 19: 3874–3882
Zhang Z, Li H, Kasmi S, Van Herck S, Deswarte K, Lambrecht BN,
Hoogenboom R, Nuhn L, De Geest BG. Angew Chem Int Ed, 2019,
2
2
1
2
2
3
58: 7866–7872
2
2
4
5
Binauld S, Stenzel MH. Chem Commun, 2013, 49: 2082
Jain R, Standley SM, Fréchet JMJ. Macromolecules, 2007, 40: 452–
37 Gibson MI, O'Reilly RK. Chem Soc Rev, 2013, 42: 7204–7213
38 Wuts, P. G. M, Greene, T. W. Greene’s Protective Groups in Organic
Synthesis. 4th ed. Hoboken: John Wiley & Sons, Inc., 2006
39 Kotke M, Schreiner PR. Tetrahedron, 2006, 62: 434–439
40 Kotke M, Schreiner PR. ChemInform, 2007, 38: 779–790
41 Deslongchamps P, Dory YL, Li S. Tetrahedron, 2000, 56: 3533–3537
42 Kasmi S, Louage B, Nuhn L, Verstraete G, Van Herck S, van Steen-
bergen MJ, Vervaet C, Hennink WE, De Geest BG. Polym Chem,
2017, 8: 6544–6557
457
2
2
6
7
Heffernan MJ, Murthy N. Bioconjugate Chem, 2005, 16: 1340–1342
Murthy N, Xu M, Schuck S, Kunisawa J, Shastri N, Fréchet JMJ. Proc
Natl Acad Sci USA, 2003, 100: 4995–5000
Shenoi RA, Narayanannair JK, Hamilton JL, Lai BFL, Horte S,
Kainthan RK, Varghese JP, Rajeev KG, Manoharan M, Kizhakkedathu
JN. J Am Chem Soc, 2012, 134: 14945–14957
2
8