# The role of polymer structure on water confinement in poly(_N_
-isopropylacrylamide) dispersions11footnotemark: 1

E. Buratti  L. Tavagnacco  M. Zanatta  E. Chiessi  S. Buoso  S. Franco  B.
Ruzicka  R. Angelini  A. Orecchini  M. Bertoldo  E. Zaccarelli
<EMAIL_ADDRESS>CNR-ISC and Department of Physics, Sapienza
University of Rome, I-00185 Roma, Italy  Department of Physics, University of
Trento, I-38123 Trento, Italy  Department of Chemical Sciences and
Technologies, University of Rome Tor Vergata, I-00133 Roma, Italy  ISOF-CNR
Area della Ricerca di Bologna, I-40129 Bologna, Italy  Department of Physics
and Geology, University of Perugia, I-06123, Perugia, Italy  Department of
Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara,
I-44121 Ferrara, Italy

###### Abstract

Poly(_N_ -isopropylacrylamide) (PNIPAM) is a synthetic polymer that is widely
studied for its thermoresponsive character. However, recent works also
reported evidence of a low temperature (protein-like) dynamical transition
around 225 K in concentrated PNIPAM suspensions, independently of the polymer
architecture, i.e., both for linear chains and for microgels. In this work, we
investigate water-polymer interactions by extensive differential scanning
calorimetry (DSC) measurements of both systems, in order to understand the
effect of the different topological structures on the solution behaviour, in
particular regarding crystallization and melting processes. In addition, we
compare protiated and deuterated microgels, in both water and deuterated
water. The DSC results are complemented by dynamic light scattering
experiments, which confirm that the selective isotopic substitution
differently affects the solution behaviour. Our findings highlight the
important role played by the polymer architecture on the solution behaviour:
indeed, microgels turn out to be more efficient confining agents, able to
avoid water crystallization in a wider concentration range with respect to
linear chains. Altogether, the present data will be valuable to interpret
future low-temperature investigations of PNIPAM dispersions, particularly by
neutron scattering experiments.

###### keywords:

water, crystallization, PNIPAM, microgels, polymer structure

††journal: -

##  1 Introduction

Poly(_N_ -isopropylacrylamide) (PNIPAM) is a thermoresponsive polymer whose
aqueous solutions show a Lower Critical Solution Temperature (LCST) around 305
K [1], where the polymer undergoes a conformational transition: below it,
chains are hydrated and dispersed in water; above it, they become insoluble,
leading to a sharp but reversible coil-to-globule transition.

Several works have highlighted that PNIPAM, due to its amphiphilic character
and the chemical analogy to polypeptides, can be used as model to mimic
protein folding and denaturation [2, 3, 4, 5]. In particular, a recent
investigation by means of elastic incoherent neutron scattering (EINS)
measurements on PNIPAM chains at different degree of hydration reported
evidence of the occurrence of a protein-like dynamical transition around 225 K
[6]. This transition was observed upon cooling PNIPAM dispersions in D2O down
to very low temperatures, where notably solvent crystallization was avoided at
high enough polymer concentration. Related works investigated the role of
polymeric architecture on this transition, which has been detected both in
linear chains and microgels [7, 8, 6]. Microgels are colloidal particles
consisting of a cross-linked polymer network, with a structure that is
typically characterized by a dense core surrounded by a loose corona [9, 10].
In microgels the analogue of LCST demixing is represented by the occurrence of
a so-called Volume Phase Transition (VPT) from a swollen to a collapsed state
[11], which is reversible and allows to tune the size of the particles, a very
appealing feature for both fundamental science [12, 13, 14] and a variety of
nano and bio-technological applications [15, 16, 17, 18]. Moreover, microgels
internal architecture appears to be particularly suitable to efficiently
confine water molecules [19, 20, 7] and avoid their crystallization down to
very low temperatures. This is important both for fundamental science, as it
could provide a way to investigate water properties in the so-called no man’s
land [21], where interesting phenomena, such as a liquid-liquid phase
transition [22], should take place and of which there is so far no direct
evidence. In addition, the understanding of the mechanism that leads to
avoided water crystallization is also relevant in biological systems [23] for
a variety of applications, ranging for example from food industry [24, 25] to
tissues and organs preservation [26]. To this aim, it appears extremely
interesting to investigate the high-concentration and low-temperature regime
of microgel suspensions, in order to better understand their interplay with
solvent crystallization. Notwithstanding the wide literature on PNIPAM, this
regime is still rather unexplored.

Differential Scanning Calorimetry (DSC) can be a very useful technique to
probe the evolution of the hydration properties of these systems and to
monitor the behaviour of bulk and hydration water. However, DSC was mostly
applied so far to study the temperature-induced phase separation of PNIPAM
aqueous solutions [3, 27, 28, 29, 30, 31, 32, 33], typically at very low
polymer concentrations and in limited temperature intervals across the LCST.
Only a scarce number of papers reported on the phase behaviour, as detected by
DSC, of PNIPAM linear chains in water [20] and of chemically cross-linked
PNIPAM hydrogels [19] in a wider range of polymer concentration, extending the
analysis to temperatures far below the water crystallization conditions.

In this work, we investigate the role of polymer structure, water content and
water-polymer interactions on the VPT as well as on the melting and
crystallization processes. To this aim, we perform extensive DSC measurements
of PNIPAM suspensions in a wide range of polymer concentration, from 1% up to
80% by weight and in a temperature interval between 193 K and 333 K. Moreover,
we study dispersions of both PNIPAM linear chains and PNIPAM microgels, to
understand the effect of the architecture on the solution behaviour and on the
capability of efficiently confining water. In order to be able to compare our
results with those obtained from neutron scattering experiments, polymer
chains and microgels were dispersed both in water and in deuterium oxide.
Since we work at rather high polymer concentrations, we carefully study the
aging of the samples, repeating the DSC measurements until reaching a
stationary state. Also, to further investigate water-polymer interactions,
properties of protiated and deuterated microgels were compared. Finally, DSC
results are complemented by dynamic light scattering (DLS) experiments in
order to properly characterize the VPT of the different samples. Our results
show that both the polymer structure and the isotope substitution have an
effect on the interactions with the solvent molecules and influence the upper
limit of polymer concentration at which water is able to crystallize. We
observe some differences also in the low-concentration regime, especially
regarding the PNIPAM conformational transition, where the deuterium
substitution plays a very relevant role.

##  2 Materials and methods

_N_ -isopropylacrylamide (NIPAM) (Sigma-Aldrich, St. Louis, MO, USA), purity
97%, and deuterated _N_ -isopropylacrylamide (d-10) (d-NIPAM) (Polymer Source,
Inc, Quebec, Canada), purity $\geq 98\%$, were purified by recrystallization
from hexane, dried under reduced pressure (0.01 mmHg) at room temperature and
stored at 253 K. N,N’-methylenebisacrylamide (BIS) (Sigma-Aldrich, St. Louis,
MO, USA), electrophoresis grade, was purified by recrystallization from
methanol, dried under reduced pressure (0.01 mmHg) at room temperature and
stored at 253 K. Sodium dodecyl sulphate (SDS), purity 98% and potassium
persulfate (KPS), purity 98% were purchased from Sigma-Aldrich (St. Louis, MO,
USA) and used as received. Poly(_N_ -isopropylacrylamide) linear chains Mw=189
kDa (Plin189) (PDI 2.88) (Polymer Source, Inc, Quebec, Canada) and Mn=40 kDa
(Plin40) (Sigma-Aldrich, St. Louis, MO, USA) were used as received. Ultrapure
water (resistivity: 18.2 MW/cm at room temperature) was obtained with Arium®
pro Ultrapure water purification Systems, Sartorius Stedim. All the other
solvents (Sigma Aldrich RP grade) were used as received. Dialysis membrane,
SpectraPor® 1, MWCO 6-8 kDa (Spectrum Laboratories, Inc., Piscataway, NJ, USA)
was soaked in distilled water for 2 h and then thoroughly rinsed before use.

###  2.1 Microgels synthesis

Two different PNIPAM microgels, a protiated and a deuterated one, were
synthesised by standard precipitation polymerization. The chemical structures
of the main repeting units is reported in Fig. 1. In the case of the non-
deuterated microgel, that we refer to as h-Pm, NIPAM (0.137 M), BIS (1.87 mM)
and SDS (7.82 mM) were solubilized in 1560 mL of ultrapure water into a 2 L
jacket reactor. The solution was deoxygenated by bubbling nitrogen for 1 h and
then heated at 343 K. Initiator KPS (2.44 mM) was dissolved in 10 mL
deoxygenated water and added to initiate the polymerization, that was left to
proceed for 4 h. Deuterated PNIPAM microgel, labelled as d-Pm, was synthesised
using the same molar concentration of reactants but substituting NIPAM with
NIPAM-d10. As the crosslinker concentration is much smaller than the monomer
one, non-deuterated BIS was used for both microgels. The crude dispersions
were purified by dialysis (MWCO 6–8 kDa) with distilled water with frequent
water change for 2 weeks, then they were concentrated by lyophilisation up to
10 wt% in H2O. To prepare samples in deuterium oxide, two cycles of
lyophilisation to dryness and redispersion in D2O (10 wt%) were performed, in
order to ensure the quantitative removal of H2O.

Figure 1: Chemical structures of the main repeating unit in the protiated (on
the left) and the deuterated (on the right) PNIPAM microgels.

###  2.2 Differential Scanning Calorimetry

Thermal analyses on PNIPAM samples, both crosslinked microgels and linear
polymers, were performed with a Perkin Elmer Pyris Diamond DSC equipped with
Intracooler III as cooling system and a DSC 8000 Perkin Elmer differential
scanning calorimeter equipped with Intracooler II as cooling system. About
10-15 mg of PNIPAM dispersions at different concentrations was analysed under
nitrogen atmosphere (20 mL/min) in hermetic sealed steel pans, to prevent
changes in concentration during the heating/cooling steps. The weight of
sample containing pans was checked after experiments and no changing in weight
was observed. Samples were prepared by pouring a dispersion at 10 wt% (either
in H2O or D2O) into the pans and then evaporating the exceeding solvent in a
desiccator, until the desired concentration was reached. The concentration was
regularly monitored by accurate weighing of the samples. Once reached the
target concentration, pans were hermetically sealed and analysed. The
measurements were carried out by cooling the system from 293 to 193 K, holding
the temperature for 3 minutes, then heating it to 333 K, holding the
temperature for 3 minutes and finally by cooling it again to 293 K. Each
cooling and heating step was carried out with a scanning rate of 5 K/min.
Additional scanning rates of 10 and 20 K/min were employed for assessing the
onset of the glass transition. Several cycles of cooling and heating were
performed on each sample in order to investigate the aging behaviour of all
detectable transitions and whether a long-time stationary state can be
reached. The details of the investigation are reported in the Supplementary
Material. In the remaining of this article, we only focus on data taken after
several thermal cycles and in long-time measurements at all concentrations,
thus avoiding the interference of aging effects. In addition, the procedure
that we used to analyse all the transitions in the thermograms, in particular
regarding the melting peak, is also described in more detail in the
Supplementary Material.

###  2.3 Dynamic Light Scattering

Dynamic Light Scattering (DLS) measurements were performed on non-deuterated
and deuterated PNIPAM microgels at concentration of 0.01% by weight in both
H2O and in D2O, in a range of temperatures between 293 K and 317 K.
Measurements were carried out using an optical setup based on a solid state
laser (100 mW) with a monochromatic ($\lambda$ = 642 nm) and polarized beam,
at a scattering angle $\theta$ = 90° corresponding to a scattering vector
$Q=0.018\,\mathrm{nm}^{-1}$, according to the relation $Q=(4\pi
n/\lambda)\sin(\theta/2)$. The hydrodynamic radius of the particles was
obtained through the Stokes-Einstein relation $R=k_{B}T/6\pi\eta D_{t}$, where
$k_{B}$ is the Boltzmann constant, $\eta$ the viscosity and $D_{t}$ the
translational diffusion coefficient, related to the relaxation time $\tau$
through the relation $\tau=1/(Q^{2}D_{t})$. The relaxation time $\tau$ was
obtained by fitting the autocorrelation function of scattered intensity
through the Kohlrausch-William-Watts expression,
$g_{2}(Q,t)=1+b[\exp(-(t/\tau)^{\beta})]^{2}$, as commonly used for colloidal
systems with low polydispersity [34], where $\beta$ is the stretching
exponent.

##  3 Results

We start by reporting a representative DSC thermogram of h-Pm microgels at 40%
weight in Fig. 2. It can be observed that, upon cooling the dispersion from
room temperature down to very low temperature (193 K), crystallization of
water occurs, as evident from the exothermic peak between the blue lines. At
lower temperatures, a glass transition appears, manifesting itself as a change
of slope (in the thermogram between orange lines), both under cooling and then
under heating. Then upon re-heating up to room temperature, the melting
(endothermic peaks between red lines) of the previously crystallized solvent
is noticed, preceded by a process of cold-crystallization (exothermic peak). A
further increase of temperature leads to the PNIPAM conformational transition
around 305 K, associated to the VPT in this case, that is shown by the
endothermic peak between green lines.

Figure 2: Thermogram of h-Pm microgels at a concentration of 40 wt% in H2O.

###  3.1 Study of the water melting

In previous works [7, 6], we observed the occurrence of a protein-like
dynamical transition in PNIPAM microgels as well as in PNIPAM linear chains at
high concentrations, above roughly 40 wt%, where the crystallization of water
was absent at low temperatures. These results are in qualitative agreement
with previous DSC measurements [19], but we aim to be a bit more quantitative
on such crystallization in order to highlight the role of the polymer
architecture on water confinement and on the suppression of crystallization.

Therefore, we now provide a careful investigation of this issue, aiming to
establish in which range of polymer concentration water crystallization is
suppressed. By means of calorimetric measurements, we can specifically obtain
quantitative information on the solvent behaviour through the analysis of its
melting peak. In Fig. 3, as an example, we report the evolution of the melting
peak as a function of polymer concentration for the non-deuterated microgels
in D2O. It can be seen that there is a shift of the main peak toward lower
temperatures by increasing concentration, associated to a decrease of the peak
area, in agreement with the lower amount of solvent in more concentrated
dispersions. In addition, for PNIPAM concentrations higher than 30 wt%, two
endothermic peaks are observed: one in the range 268 – 274 K and the other
around 276 K. The latter peak is always found approximately at this
temperature value for samples in D2O, while it is located around 274 K for
samples in H2O.

Figure 3: Melting peaks of h-Pm in D2O for samples in the range of
concentration 10 – 70 wt%. Thermograms are normalized to the mass of
dispersion.

To better visualize the evolution of the $T$-dependence of these melting
peaks, we plot in Fig. 4 the temperatures associated to the maximum of the
calorimetric peaks as a function of polymer concentration for all investigated
structures and dispersions. All studied samples point to the fact that there
is evidence of melting of two different types of water, in agreement with
previous works [35]. Indeed, the peak at higher temperature (open symbols) can
be associated to the so-called “free water” (bulk-like water), since it has a
melting point roughly equal to the one of the pure solvent ($\sim 274$ K for
H2O and $\sim 276$ K for D2O) and it is found to be quite independent of
concentration. On the other hand, the peak observed at lower temperature
(closed symbols) is much more affected by the presence of the polymer and its
melting temperature shifts toward lower values by increasing PNIPAM
concentration. For these reasons, it appears to be associated to crystallizing
hydration water, which has previously been referred as “freezable-bound water”
[35]. The onset of these two types of water is general and happens for all the
studied samples, regardless of the polymer architecture and type of solvent.
We further notice in Fig. 3 that, only for concentrations around 30-40 wt%, a
third, additional peak also arises, of very small intensity, at even lower
temperatures. This peak is present in this range of concentrations for all
different samples, thus appearing a robust feature of the thermograms. This
could be tentatively associated to yet another type of water affected by the
polymer interaction [36], for example located in a further proximal domain,
possible only at intermediate concentrations. At higher concentrations, the
peak disappears since in that case it is not possible to coordinate water
molecules in the more distant coordination shells. A second hypothesis is that
this peak may be due to small crystals of confined water. This might explain
the behaviour of the melting temperature, which is well known to decrease with
crystal size, due to the predominant effect of the excess of energy at the
crystal surface.

Figure 4: Melting temperature of the “free water” (open symbols) and of the
“freezable-bound water” (closed symbols) for all studied polymer-solvent
combinations as a function of PNIPAM concentration. Lines are guides to the
eye. The melting temperature of the third peak appearing only at intermediate
concentrations is represented by crosses. The vertical dotted lines represent
the lower limit above which solvent crystallization is completely absent.
Errors on temperature values are estimated to be of the order of 0.5 K. Error
bars are reported with the data but they are not clearly visible in the scale
of the figure.

While the temperature of the melting peaks is relatively unaffected by the
polymer structure, we find a clear indication that this plays an important
role in the value of the critical concentration above which melting was no
longer detected, as also reported in Fig. 4. We note that such a critical
value is significantly lower for PNIPAM microgels (around 60 wt%) than for
linear chains (70-80 wt%), suggesting that the topological constraint induced
by the presence of crosslinks in the network acts against proper water-water
hydrogen bonds rearrangements and thus crystallization. In the case of Plin189
and Plin40, instead, the water molecules and the linear chains are more free
to move and rearrange and thus the critical value is shifted to higher
concentrations. Interestingly, we do not observe significant differences in
the concentration threshold for chains at the two studied molecular weights,
in agreement with previous studies [19]. Similarly, no significant differences
in the value of the cystallization limit are observed on changing solvent from
water to deuterium oxide and/or using deuterated microgels.

Figure 5: Melting enthalpy of the total peak as a function of polymer
concentration for all studied samples. Green and blue lines are the linear
regression of the data of Plin189 in H2O and h-Pm in H2O, respectively.
Estimated slopes are -4.6 $\pm$ 0.3 and -5.9 $\pm$ 0.3, respectively. The
percentage error on the enthalpy values is estimated to be around 2.5%. Error
bars are reported with the data but they are not clearly visible in the scale
of the figure.

We now turn to examine the behaviour of the peak area and of the associated
melting enthalpy, which is found to decrease with polymer concentration, as
expected. In Fig. 5, the melting enthalpy of the entire peak is reported for
the dispersions at different concentrations. For the most dilute conditions,
enthalpy is around 300 J/g, close to the value of the pure solvents, as also
previously confirmed in Ref. [19]. Then a roughly linear decrease is observed,
that appears to be steeper for microgels than for linear chains, as we can see
in the figure from the comparison of the linear fits of Plin189 and h-Pm both
in H2O, until it reaches zero at polymer concentration around 60 or 75 wt%,
respectively.

In order to further analyse the interactions of the microgels and of the
linear chains with the “free” and “freezable-bound” water , we developed a
procedure to separate the areas of the corresponding peaks, as described in
more detail in the Supplementary Material.

The resulting fraction of melting enthalpy associated to each peak is reported
in Fig. 6. Data are normalized to the results at the most dilute investigated
concentration (1 wt%). We observe that free water (open symbols) is the only
type of water present at low concentration, while the onset of freezable-bound
water arises for all samples around 30-40 wt%. Above this value, the enthalpy
associated to free water rapidly drops while that related to freezing
hydration water remains detectable up to the high concentration limit of
crystallization.

Figure 6: Normalized melting enthalpy of free water (open symbols) and
freezable-bound water (closed symbols) for each investigated polymer-solvent
case. Lines are guides to the eye. The melting enthalpy associated to the
third peak occurring at intermediate concentrations is represented by the
crosses. The area of the peak of 1% samples is taken as the reference case at
100%.

While we have examined the tendency to crystallize, we have not been
quantitative on this aspect yet. The degree of crystallinity $\chi_{c}$ in the
dispersion can be estimated through the following equation [37]:

$\chi_{c}\,(\mathrm{wt\%})=\frac{\Delta H_{m}}{\Delta
H^{\circ}_{m}}\frac{1}{\left(1-\frac{C_{w}^{\mathrm{PNIPAM}}}{100}\right)}100,$
(1)

where $\Delta{H^{\circ}_{m}}$ is the standard fusion enthalpy either of bulk
H2O at 273 K, that is 333 J/g, or of bulk D2O at 276.7 K, that is 340.7 J/g,
(1 - $\frac{C^{\mathrm{PNIPAM}}_{w}}{100}$) is the weight fraction of H2O or
D2O in the sample and $\Delta{H_{m}}$ (J/gsample) is the enthalpy associated
with the overall peak, that is the sum of “free water” and “freezable-bound
water”. It is important to note that the values of $\chi_{c}$ obtained in this
way are entirely attributable to the solvent, since poly(_N_
-isopropylacrylamide) is an amorphous polymer, and hence by this quantity we
can directly compare results for different polymer structures and solvent
content.

Figure 7: Crystallization degree, $\chi_{c}$, as a function of polymer
concentration for each sample, calculated from Equation 1.

Fig. 7 shows that similar crystallinity degrees are found at high solvent
content for all the samples, while clear differences are present upon
increasing PNIPAM concentration above 40 wt%. In agreement with the
information obtained from the melting temperature, we find that $\chi_{c}$ is
significantly larger for linear chains than for microgels, indicating that
crystallization is facilitated by the higher mobility of the chains. Results
for the two different molecular weights of linear PNIPAM are similar to each
other, in agreement with previous work [19]. However, a slight difference
between the two can be noticed at high concentration, where the value of the
crystallinity for Plin189 is higher than for Plin40. Since the two samples
only differ in their molecular weight and thus in the concentration of chain-
ends, which is higher for the smaller chains with respect to the bigger ones,
this result suggests a possible role of chain-ends in the reduction of water
crystallization.

Moreover, comparing crystallinity at the same polymer concentration, it can be
observed that the $\chi_{c}$ values in deuterium oxide are always larger than
the corresponding ones in water. This happens for linear chains and also for
microgels, both non-deuterated and deuterated ones, indicating a higher
affinity of the polymer, regardless of the structure, with water in the
investigated low temperature/ high concentration range. By comparing the
effect of polymer deuteration on crystallization, it seems that h-Pm have a
larger suppressing effect than d-Pm, both in water and deuterium oxide,
suggesting that the interactions of the non-deuterated microgels with solvents
are higher and affect a larger number of solvent molecules as compared to
deuterated microgels.

From these data, we can confirm that melting completely disappears for
microgel samples at concentration above 60 wt%. However, already at a
concentration above 40 wt%, we find that the value of $\chi_{c}$ is of the
order of just a few percent. This may well explain the fact that in previous
EINS measurements [7], the presence of D2O crystals was already not detectable
at 43 wt% for microgels. The same considerations apply for linear chains,
albeit at slightly larger concentration values [6].

###  3.2 Study of the glass transition

The glass transition of concentrated dispersions of PNIPAM linear chains and
microgels was investigated by performing DSC measurements with cooling and
heating steps at 5, 10 and 20 K/min. In Fig. 8 the glass transition
temperature, $T_{g}$, measured in the heating step of the thermograms
collected at 5 K/min, is reported for all samples where this was detected. For
samples in water, the glass transition is observed only for concentrations
equal and above 40 wt%, while for samples in deuterium oxide this happens
above 50 wt%. However, we note that we could not find a clear evidence of the
glass transition for all values of polymer concentrations measured between 40
and 80 wt%. To this aim, we compared thermograms at different scanning rates
and reported data points only for the conditions where this was found to be
robust. A reason why it appears to be difficult to locate the glass transition
at intermediate concentrations is that this may be hidden by the concomitant
melting or LCST/VPT peak. Fig. 8 also includes a comparison with previous
results [19] and with the calculated concentration dependence of $T_{g}$ for
miscible binary systems of PNIPAM in H2O and D2O, resulting by using the
values of the glass transition temperatures for the pure solvent and PNIPAM.
Overall, we find a good agreement between experimental points and such
theoretical predictions at high concentrations, respectively, i.e., above 60
wt%, while at lower concentrations there are deviations towards higher
temperatures in the experimental data, as also found in Ref. [19]. Such
deviation occurs when free water crystallization dominates the thermograms.
Indeed, the mixing rule is based on the assumption that water is evenly
distributed in the system. However, when a part of water crystallizes, such a
condition is not valid. Therefore, the observed glass transition is the one of
the amorphous phase whose composition levels off for Cw below 40%, when free
water starts to crystallize. In general, the difficulty to precisely detect a
glass transition temperature in the whole investigated range of concentrations
within our current analysis prevents us from assessing more quantitative
statements about the dependence of $T_{g}$ on the specific polymer
architecture and on the influence of isotopic substitution. It would be thus
interesting to address this point in more detail in the future with more
sensitive calorimetric protocols.

Figure 8: Glass transition temperature, $T_{g}$, as a function of polymer
concentration for each sample. Grey stars are data taken from [19]. The solid
and dashed lines represent the concentration dependence of $T_{g}$ for PNIPAM
in H2O and D2O, respectively, calculated using the Fox-equation [38]:
$1/T_{g}=w_{1}/T_{g1}+w_{2}/T_{g2}$, where $w_{1}$ and $T_{g1}$ are the weight
fraction and glass transition temperature of pure PNIPAM, $w_{2}$ and $T_{g2}$
the weight fraction and glass transition temperature of pure solvent (either
H2O or D2O) and $T_{g}$ the glass transition temperature of the dispersion.
Errors on temperature values are estimated to be of the order of 0.5 K.

###  3.3 Study of the Volume Phase transition / LCST

After having examined the melting processes, we now focus on the behaviour of
the PNIPAM coil-to-globule (for linear chains) and volume phase (for
microgels) transition. In Fig. S3, an example of the evolution of the peak as
a function of polymer concentration for the non-deuterated microgels in D2O is
reported. Some differences among the kind of structures investigated and their
interaction with solvents can be observed as reported in Fig. 9, where the
onset temperature, $T_{onset}$, which is the temperature at the beginning of
the transition, is reported for all the samples as a function of polymer
concentration.

The $T$-dependence is found to be similar for the LCST temperature of Plin189
and for the VPT temperature of h-Pm in H2O: they are found to be just shifted
at a lower value for linear chains with respect to microgels. However, in both
cases we observe a minimum value of $T_{onset}$ occurring at around 40-50 wt%,
followed by an increase at higher concentration, in good agreement with
previous works [19, 39]. A different scenario occurs in D2O: for microgels,
the behaviour of $T_{onset}$ with concentration has the same trend as in H2O,
although at high concentration it does not rise as much as in water; for
linear chains, instead, the minimum is almost not observable. Indeed, an
almost linear decrease of Tonset is evident for both Plin189 and Plin40 in D2O
up to concentration of 70 wt%, above which the LCST is no longer detectable.
Turning to the deuterated microgels, it seems that $T_{onset}$ only decreases
with concentration, with a more pronounced reduction in D2O than in H2O, in
analogy with what observed for h-Pm and linear chains in the two different
solvents.

Finally, comparing the behaviour between the two solvents, it appears that at
low concentrations the transition temperature is always higher in deuterium
oxide than in water, while increasing concentration above 40% this trend is
inverted, with values in D2O always lower than those in H2O. The higher
transition temperature detected for concentrated dispersions in H2O indicates
a greater affinity of PNIPAM for H2O, as compared to D2O, in the high
concentration regime. A similar conclusion was drawn in a previous study of
H2O/D2O exchange in dense and homogeneous PNIPAM thin films, whose hydration
levels were below 35% (v/v) [40]. In dilute regime an opposite trend of
solvent affinity is found, with affinity being higher in D2O with respect to
H2O, in agreement with previous calorimetric experiments for PNIPAM chains at
0.1 wt% [29]. In addition, for very low concentrations (1-10 wt%), we observe
that d-Pm microgels show values of $T_{onset}$ that are significantly higher
($\sim$ 308-310 K) than those of all other samples, both in H2O and in D2O.

For completeness, the enthalpies associated to the transition are reported as
function of polymer concentration in Fig. S4. The values reach a maximum
around 30-40 wt% for linear chains and microgels. However, the enthalpy
associated with the transition of the linear chains is slightly higher than
the one of the microgels, as well as the one in D2O as compared to the one in
H2O.

Figure 9: Onset temperature $T_{onset}$ of the PNIPAM coil-to-globule (for
Plin189 and Plin40) and volume phase (for h-Pm and d-Pm) transition, as a
function of polymer concentration, for all investigated samples. Errors on
temperature values are estimated to be of the order of 0.5 K.

###  3.4 Swelling behaviour of PNIPAM microgels

In order to better quantify the differences induced by the specific solvent
and by the nature of the microgels (deuterated vs non-deuterated), we perform
dynamic light scattering measurements on the microgels in dilute conditions
both in H2O and D2O. The corresponding swelling curves are reported in Fig. 10
as a function of temperature. The experimental data were fitted with a
Boltzmann equation [41, 42] and the derivatives of each function are shown in
the bottom of the figure. The peaks of these derivatives represent the
estimated VPT temperature at which microgels change from a highly hydrated
swollen state into a partially dehydrated, collapsed state.

Figure 10: (Top) Swelling curve of non-deuterated and deuterated PNIPAM
microgels in H2O and D2O. (Bottom) Derivative of the fit of the experimental
data in the upper part. The VPT temperatures of each sample are also reported.
Data refer to microgel dispersions at a concentration of 0.01 wt%.

We find that the value of VPT temperature (VPTT) is considerably affected by
the isotope substitution: in the case of h-Pm in H2O, the VPTT is observed at
304 K, around the classic value, while we find an increase of about two
degrees both when deuterium is added to the polymer chains, i.e., d-Pm in H2O,
or to the solvent, i.e., h-Pm in D2O. Namely, we find 306.5 and 306.7 K,
respectively, for these two cases. These results are in good agreement with
previous works on microgels in D2O [43, 42]. The higher VPTT value of h-Pm in
D2O, with respect to H2O, is consistent with the increased transition
temperature detected for this system in the DSC experiments at the lowest
polymer concentrations in D2O, as compared to H2O. A similar isotopic effect
of the solvent was also reported for the VPTT of macroscopic PNIPAM hydrogels
at low degree of crosslinking [44], where the higher stability of the
hydrogen-bonding interaction between the PNIPAM amide groups and the deuterium
oxide molecules are ascribed to the lower vibrational energy of intermolecular
hydrogen bond in D2O with respect to H2O due to the heavier mass [45].

The increase of LCST or VPTT in highly hydrated PNIPAM systems, detected
moving from the protiated to deuterated isotopologues, can be instead ascribed
to the lower lipophilicity of deuterated aliphatic groups as compared to the
non-deuterated ones, because of the reduced vibrations of deuterons with
respect to protons [46, 47], which leads to a stabilization of the hydrated
states. Similarly, the deuteration of non-exchangeable protons in globular
proteins was found to reduce their denaturation temperature of few degrees
[48, 49, 50], both in H2O and D2O [51], confirming a deuteration-induced
decrease in stability of globular, collapsed states. Differently, for a few
proteins in the native isotopic state, an increase of the denaturation
temperature in D2O was observed [52, 53, 54, 55], which can be explained with
the higher strength of inter-amide hydrogen bonds, when changing H with D
atoms [56, 57]. However, PNIPAM collapsed states still remain partially
hydrated [11], thus the contribution of inter-residue H bonds to its
conformational transition is weak, becoming relevant only for highly
concentrated dispersions, as also shown by the present calorimetric results.
As a consequence, the prevailing factor in diluted aqueous environments moving
from H2O to D2O is the reinforcement of polymer-solvent hydrogen bonds,
leading to a stabilization of the more hydrated/swollen states. When both the
microgel and the solvent are deuterated, i.e., d-Pm in D2O, an additional
shift of two degrees is observed, to even higher values (308.2 K). While there
are a few studies that also report the shift for h-Pm in D2O [39, 58, 59, 60,
61] and for d-Pm in H2O [62, 63, 40, 64], we are not aware of any previous
report of this effect. A tentative explanation of this intriguing, new result
is that the combination of the two effects discussed above sums up, giving
rise to a “double” shift of the transition temperature. It is also worth
noting that the transition is sharper in D2O than in H2O for both h-Pm and
d-Pm, as can be seen from the peak broadness of the derivative, that is
especially narrow for the non-deuterated polymer in D2O. This is in good
agreement with other works on PNIPAM-based materials [65, 66, 44, 67]. The
shape of the transition for h-Pm and d-Pm in H2O is instead almost identical.

Finally, a comment is due on the swelling degree, $\alpha$, that is defined as
the ratio between the particle radius in the swollen state (at 293 K) and that
in the collapsed state (at 313 or 317 K). This quantity also seems to be
affected by either the isotope or the solvent substitution. In fact, while
h-Pm microgels have a comparable size both in H2O and D2O in the collapsed
form, this does not apply to the swollen one, leading to a change in the
swelling degree from roughly 2 to 2.5, respectively. A different scenario
occurs for d-Pm, where the collapsed state is larger in D2O, while the swollen
ones are similar. This leads to $\alpha$ roughly equal to 1.7 in H2O and 1.4
in D2O. However, these values are significantly lower than the ones of h-Pm in
either H2O or D2O. Altogether these features suggest a lower energy of the
polymer-polymer interactions in the case of d-Pm with respect to h-Pm and even
a lower difference (polymer-polymer, polymer-solvent) in the case of the
deuterated microgels.

##  4 Conclusions

In this work, we investigated how the polymer architecture influences water
behaviour in concentrated samples of poly(_N_ -isopropylacrylamide): two
PNIPAM linear polymers at two different molecular weights and two weakly
crosslinked microgels, one of which synthesised from a deuterated monomer. The
aim was to provide a detailed investigation of water crystallization in poorly
hydrated PNIPAM suspensions and in particular to study the possible effects of
isotope substitution. Indeed, recent elastic neutron scattering measurements
pointed out that hydrated PNIPAM powders undergo a protein-like dynamical
transition [8, 7] and suggest that hydration water does not crystallize even
at polymer concentrations as low as 50 wt%. Since the protein-like dynamical
transition is often considered to be driven by the atomic dynamics of the
solvent [68, 69], independent and reliable information about the amorphous or
crystalline state of hydration water is extremely valuable for a robust
interpretation of neutron data. Moreover, the subject of avoided
crystallization in confined water is per se a long standing problem in the
physics of water itself [70, 22]. In addition, since the previous works were
mainly based on neutron scattering experiments in D2O environment, we also
compare the behaviour of PNIPAM samples in H2O and D2O, in order to highlight
possible differences between the two solvents. Through calorimetric analysis
in a wide range of temperatures and polymer concentrations, we monitored, in
particular, the behaviour of the melting peak and of the one corresponding to
the PNIPAM thermoresponsive transition (LCST and VPT for linear chains and
microgels, respectively). We also, when possible, paid attention to the
occurrence of a glass transition in the samples.

Overall, we found strong indications that PNIPAM microgels are more efficient
in suppressing water crystallization with respect to linear chains, since they
prevent water crystallization at lower polymer concentration (60% against 75%,
respectively). Moreover, in this high-concentration regime (50-80 wt%), we
observed that solvent-polymer interactions are preferred to those between
solvent molecules to a larger extent when the solvent is H2O, as shown by the
higher degree of crystallinity obtained in D2O than in H2O. This greater
affinity with aqueous solvent in concentrated dispersions is also shown in the
PNIPAM conformational transition: indeed, the transition temperature above
$\geq 40$ wt% polymer concentration is found to be higher for samples in H2O
than in D2O, indicating that the polymer finds water a “better” solvent than
deuterium oxide, as proposed in [40]. This may ascribed to the fact that, at
high polymer concentrations, the contribution of polymer-polymer H-bonds in
the transition become more important. In D2O the amides are ND(CO), so that
the inter-residue H-bonds are stronger than in H2O, where they occur between
NH(CO) groups. Thus, it is more convenient to increase the number of polymer-
polymer H-bonds in D2O than in H2O, leading to the anticipation of the
transition. Instead, in the low-concentration regime, this aspect is reversed,
since the transition happens at lower temperature in H2O, confirming that the
polymer-D2O interactions are stronger than the polymer-H2O interactions in
this regime, as also supported by the swelling behaviour and DLS measurements
of the microgels in very dilute conditions.

Finally, we analysed the isotope substitution not only by changing the
solvent, but also investigating a deuterated microgel. We found that, in the
low-concentration regime, the use of either deuterated particles in H2O or
non-deuterated microgels in D2O shifts the VPT temperature by roughly two
degrees, as previously observed in several works [39, 58, 59, 62], while the
consideration of deuterated particles in D2O induces an unexpected “double”
shift of the VPT temperature, occurring roughly four degrees later than the
standard one. The increase of the VPTT from h-Pm to d-Pm in D2O remains to be
clarified. We tentatively attribute this feature to the effect of PNIPAM
hydrophobic groups. In particular, the protiated hydrophobic groups are more
lipophilic (and therefore less affine to water, be it H2O or D2O) than the
deuterated ones. Thus, the increase of contacts between hydrophobic groups
occurring at the VPT is enthalpically more advantageous for h-Pm than for
d-Pm, even in D2O, as it is in H2O, leading to a lower VPTT. Overall the
double VPTT-increasing effect observed for non-deuterated microgels in D2O may
thus be due to two separate contributions, arising from different parts of the
polymer: (i) an effect arising from the hydrophilic groups (amides) and (ii)
an effect arising from hydrophobic groups. In the first case, going from H2O
to D2O, the amide-water hydrogen bonds are strengthened, so that a loss of
water is more disadvantageous in D2O, which increases the transition $\Delta
H$ and thus the VPTT, with the phenomenon being identical for h-Pm and d-Pm.
In the second case, going from h-Pm to d-Pm, the interactions between
aliphatic groups are weakened, so that so that the VPT, leading to an increase
of such interactions, is less advantageous for d-Pm than for h-Pm (regardless
of whether this happens in H2O or D2O). These factors may thus explain the
additivity of the “double” shift of the VPT temperature.

We believe that our results will be valuable to correctly interpret past and
future neutron scattering measurements of these highly concentrated samples,
particularly to address the role of solvent crystallization and to
differentiate the effects due to the use of D2O vs H2O. Given the relevance of
the low-temperature dynamical transition in biological samples and the
shortage of similar calorimetric studies for these systems [71], our work
could be important to address these issues also for protein dispersions,
shedding further light on the role of the solvent in this debated topic [8].

##  5 Acknowledgement

We acknowledge financial support from MIUR (FARE project R16XLE2X3L, SOFTART),
from the European Research Council (ERC Consolidator Grant 681597, MIMIC) and
from Regione Lazio (through L.R. 13/08 Progetto Gruppo di Ricerca MICROARTE n.
prot. A0375-2020-36515).

## References

  * [1] A. Halperin, M. Kröger, F. M. Winnik, Poly(_N_ -isopropylacrylamide) phase diagrams: Fifty years of research, Angewandte Chemie International Edition 54 (51) (2015) 15342–15367.  doi:10.1002/anie.201506663.
  * [2] E. I. Tiktopulo, V. N. Uversky, V. B. Lushchik, S. I. Klenin, V. E. Bychkova, O. B. Ptitsyn, “Domain” coil-globule transition in homopolymers, Macromolecules 28 (22) (1995) 7519–7524.  doi:10.1021/ma00126a032.
  * [3] S. Fujishige, K. Kubota, I. Ando, Phase transition of aqueous solutions of poly(_N_ -isopropylacrylamide) and poly(_N_ -isopropylmethacrylamide), The Journal of Physical Chemistry 93 (8) (1989) 3311–3313.  doi:10.1021/j100345a085.
  * [4] G. Graziano, On the temperature-induced coil to globule transition of poly-_N_ -isopropylacrylamide in dilute aqueous solutions, International Journal of Biological Macromolecules 27 (1) (2000) 89–97.  doi:10.1016/s0141-8130(99)00122-1.
  * [5] B. P. Rosi, L. Tavagnacco, L. Comez, P. Sassi, M. Ricci, E. Buratti, M. Bertoldo, C. Petrillo, E. Zaccarelli, E. Chiessi, et al., Thermoresponsivity of poly(_N_ -isopropylacrylamide) microgels in water-trehalose solution and its relation to protein behavior, Journal of Colloid and Interface Science 604 (2021) 705–718.  doi:10.1016/j.jcis.2021.07.006.
  * [6] L. Tavagnacco, M. Zanatta, E. Buratti, B. Rosi, B. Frick, F. Natali, J. Ollivier, E. Chiessi, M. Bertoldo, E. Zaccarelli, et al., Protein-like dynamical transition of hydrated polymer chains, Physical Review Research 3 (1) (2021) 013191.  doi:10.1103/PhysRevResearch.3.013191.
  * [7] M. Zanatta, L. Tavagnacco, E. Buratti, M. Bertoldo, F. Natali, E. Chiessi, A. Orecchini, E. Zaccarelli, Evidence of a low-temperature dynamical transition in concentrated microgels, Science Advances 4 (9) (2018) eaat5895.  doi:10.1126/sciadv.aat5895.
  * [8] L. Tavagnacco, E. Chiessi, M. Zanatta, A. Orecchini, E. Zaccarelli, Water–polymer coupling induces a dynamical transition in microgels, The Journal of Physical Chemistry Letters 10 (4) (2019) 870–876.  doi:10.1021/acs.jpclett.9b00190.
  * [9] A. Fernandez-Nieves, H. Wyss, J. Mattsson, D. Weitz (Eds.), Microgel suspensions: fundamentals and applications, Wiley-VCH Verlag, Germany, 2011.  doi:10.1002/9783527632992.
  * [10] M. Stieger, W. Richtering, J. S. Pedersen, P. Lindner, Small-angle neutron scattering study of structural changes in temperature sensitive microgel colloids, The Journal of Chemical Physics 120 (13) (2004) 6197–6206.  doi:10.1063/1.1665752.
  * [11] R. Pelton, Poly(_N_ -isopropylacrylamide)(PNIPAM) is never hydrophobic, Journal of Colloid and Interface Science 348 (2) (2010) 673–674.  doi:10.1016/j.jcis.2010.05.034.
  * [12] P. J. Yunker, K. Chen, M. D. Gratale, M. A. Lohr, T. Still, A. Yodh, Physics in ordered and disordered colloidal matter composed of poly(_N_ -isopropylacrylamide) microgel particles, Reports on Progress in Physics 77 (5) (2014) 056601.  doi:10.1088/0034-4885/77/5/056601.
  * [13] L. Rovigatti, N. Gnan, L. Tavagnacco, A. J. Moreno, E. Zaccarelli, Numerical modelling of non-ionic microgels: an overview, Soft Matter 15 (6) (2019) 1108–1119.  doi:10.1039/C8SM02089B.
  * [14] J. Brijitta, P. Schurtenberger, Responsive hydrogel colloids: Structure, interactions, phase behavior, and equilibrium and nonequilibrium transitions of microgel dispersions, Current Opinion in Colloid & Interface Science 40 (2019) 87–103.  doi:10.1016/j.cocis.2019.02.005.
  * [15] J. K. Oh, R. Drumright, D. J. Siegwart, K. Matyjaszewski, The development of microgels/nanogels for drug delivery applications, Progress in Polymer Science 33 (4) (2008) 448–477.  doi:10.1016/j.progpolymsci.2008.01.002.
  * [16] A. Fernández-Barbero, I. J. Suárez, B. Sierra-Martín, A. Fernández-Nieves, F. J. de Las Nieves, M. Marquez, J. Rubio-Retama, E. López-Cabarcos, Gels and microgels for nanotechnological applications, Advances in Colloid and Interface Science 147 (2009) 88–108.  doi:10.1016/j.cis.2008.12.004.
  * [17] M. Karg, A. Pich, T. Hellweg, T. Hoare, L. A. Lyon, J. Crassous, D. Suzuki, R. A. Gumerov, S. Schneider, I. I. Potemkin, et al., Nanogels and microgels: From model colloids to applications, recent developments, and future trends, Langmuir 35 (19) (2019) 6231–6255.  doi:10.1021/acs.langmuir.8b04304.
  * [18] M. A. C. Stuart, W. T. Huck, J. Genzer, M. Müller, C. Ober, M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M. Urban, et al., Emerging applications of stimuli-responsive polymer materials, Nature Materials 9 (2) (2010) 101–113.  doi:10.1038/nmat2614.
  * [19] F. Afroze, E. Nies, H. Berghmans, Phase transitions in the system poly(_N_ -isopropylacrylamide)/water and swelling behaviour of the corresponding networks, Journal of Molecular Structure 554 (1) (2000) 55–68.  doi:10.1016/S0022-2860(00)00559-7.
  * [20] K. Van Durme, G. Van Assche, B. Van Mele, Kinetics of demixing and remixing in poly(_N_ -isopropylacrylamide)/water studied by modulated temperature DSC, Macromolecules 37 (25) (2004) 9596–9605.  doi:10.1021/ma048472b.
  * [21] O. Mishima, H. E. Stanley, The relationship between liquid, supercooled and glassy water, Nature 396 (6709) (1998) 329–335.  doi:10.1038/24540.
  * [22] P. G. Debenedetti, F. Sciortino, G. H. Zerze, Second critical point in two realistic models of water, Science 369 (6501) (2020) 289–292.  doi:10.1126/science.abb9796.
  * [23] C. A. Knight, Adding to the antifreeze agenda, Nature 406 (6793) (2000) 249–251.  doi:10.1038/35018671.
  * [24] H. Kiani, D.-W. Sun, Water crystallization and its importance to freezing of foods: A review, Trends in Food Science & Technology 22 (8) (2011) 407–426.  doi:10.1016/j.tifs.2011.04.011.
  * [25] T. Kang, Y. You, S. Jun, Supercooling preservation technology in food and biological samples: A review focused on electric and magnetic field applications, Food science and biotechnology 29 (3) (2020) 303–321.  doi:10.1007/s10068-020-00750-6.
  * [26] R. P. Tas, V. Sampaio-Pinto, T. Wennekes, L. W. van Laake, I. K. Voets, From the freezer to the clinic: Antifreeze proteins in the preservation of cells, tissues, and organs, EMBO reports 22 (3) (2021) e52162.  doi:10.15252/embr.202052162.
  * [27] E. Tiktopulo, V. Bychkova, J. Ricka, O. Ptitsyn, Cooperativity of the coil-globule transition in a homopolymer: Microcalorimetric study of poly(_N_ -isopropylacrylamide), Macromolecules 27 (10) (1994) 2879–2882.  doi:10.1021/ma00088a031.
  * [28] H. G. Schild, D. A. Tirrell, Microcalorimetric detection of lower critical solution temperatures in aqueous polymer solutions, Journal of Physical Chemistry 94 (10) (1990) 4352–4356.  doi:10.1021/j100373a088.
  * [29] P. Kujawa, F. M. Winnik, Volumetric studies of aqueous polymer solutions using pressure perturbation calorimetry: a new look at the temperature-induced phase transition of poly(_N_ -isopropylacrylamide) in water and D2O, Macromolecules 34 (12) (2001) 4130–4135.  doi:10.1021/ma002082h.
  * [30] Y. Ding, G. Zhang, Microcalorimetric investigation on association and dissolution of poly(_N_ -isopropylacrylamide) chains in semidilute solutions, Macromolecules 39 (26) (2006) 9654–9657.  doi:10.1021/ma062269u.
  * [31] X. Qiu, T. Koga, F. Tanaka, F. M. Winnik, New insights into the effects of molecular weight and end group on the temperature-induced phase transition of poly(_N_ -isopropylacrylamide) in water, Science China Chemistry 56 (1) (2013) 56–64.  doi:10.1007/s11426-012-4781-9.
  * [32] S. Sun, P. Wu, W. Zhang, W. Zhang, X. Zhu, Effect of structural constraint on dynamic self-assembly behavior of PNIPAM-based nonlinear multihydrophilic block copolymers, Soft Matter 9 (6) (2013) 1807–1816.  doi:10.1039/C2SM27183D.
  * [33] I. Bischofberger, D. Calzolari, P. De Los Rios, I. Jelezarov, V. Trappe, Hydrophobic hydration of poly-_N_ -isopropylacrylamide: a matter of the mean energetic state of water, Scientific Reports 4 (1) (2014) 1–7.  doi:10.1038/srep04377.
  * [34] V. Nigro, R. Angelini, B. Rosi, M. Bertoldo, E. Buratti, S. Casciardi, S. Sennato, B. Ruzicka, Study of network composition in interpenetrating polymer networks of poly (n isopropylacrylamide) microgels: The role of poly (acrylic acid), Journal of colloid and interface science 545 (2019) 210–219.  doi:10.1016/j.jcis.2019.03.004.
  * [35] Z. Ping, Q. Nguyen, S. Chen, J. Zhou, Y. Ding, States of water in different hydrophilic polymers—DSC and FTIR studies, Polymer 42 (20) (2001) 8461–8467.  doi:10.1016/S0032-3861(01)00358-5.
  * [36] T. Hatakeyma, H. Kasuga, M. Tanaka, H. Hatakeyama, Cold crystallization of poly(ethylene glycol)–water systems, Thermochimica Acta 465 (1-2) (2007) 59–66.  doi:10.1016/j.tca.2007.09.005.
  * [37] Y. Kong, J. Hay, The measurement of the crystallinity of polymers by DSC, Polymer 43 (14) (2002) 3873–3878.  doi:10.1016/S0032-3861(02)00235-5.
  * [38] T. G. Fox, Bulletin of the American Physical Society 1 (1956) 123.
  * [39] M. Zanatta, L. Tavagnacco, E. Buratti, E. Chiessi, F. Natali, M. Bertoldo, A. Orecchini, E. Zaccarelli, Atomic scale investigation of the volume phase transition in concentrated PNIPAM microgels, The Journal of Chemical Physics 152 (20) (2020) 204904.  doi:10.1063/5.0007112.
  * [40] T. Widmann, L. P. Kreuzer, N. Hohn, L. Bießmann, K. Wang, S. Rinner, J.-F. Moulin, A. J. Schmid, Y. Hannappel, O. Wrede, et al., Hydration and solvent exchange induced swelling and deswelling of homogeneous poly(_N_ -isopropylacrylamide) microgel thin films, Langmuir 35 (49) (2019) 16341–16352.  doi:10.1021/acs.langmuir.9b03104.
  * [41] A. L. Navarro-Verdugo, F. M. Goycoolea, G. Romero-Meléndez, I. Higuera-Ciapara, W. Argüelles-Monal, A modified Boltzmann sigmoidal model for the phase transition of smart gels, Soft Matter 7 (12) (2011) 5847–5853.  doi:10.1039/C1SM05252G.
  * [42] M. Cors, L. Wiehemeier, J. Oberdisse, T. Hellweg, Deuteration-induced volume phase transition temperature shift of PNIPMAM microgels, Polymers 11 (4) (2019) 620.  doi:10.3390/polym11040620.
  * [43] S. Nöjd, P. Holmqvist, N. Boon, M. Obiols-Rabasa, P. S. Mohanty, R. Schweins, P. Schurtenberger, Deswelling behaviour of ionic microgel particles from low to ultra-high densities, Soft Matter 14 (20) (2018) 4150–4159.  doi:10.1039/C8SM00390D.
  * [44] H. Shirota, N. Endo, K. Horie, Volume phase transition of polymer gel in water and heavy water, Chemical Physics 238 (3) (1998) 487–494.  doi:10.1016/S0301-0104(98)00340-1.
  * [45] G. Nemethy, H. A. Scheraga, Structure of water and hydrophobic bonding in proteins. IV. The thermodynamic properties of liquid deuterium oxide, The Journal of Chemical Physics 41 (3) (1964) 680–689.  doi:10.1063/1.1725946.
  * [46] M. Turowski, N. Yamakawa, J. Meller, K. Kimata, T. Ikegami, K. Hosoya, N. Tanaka, E. R. Thornton, Deuterium isotope effects on hydrophobic interactions: the importance of dispersion interactions in the hydrophobic phase, Journal of the American Chemical Society 125 (45) (2003) 13836–13849.  doi:10.1021/ja036006g.
  * [47] D. Wade, Deuterium isotope effects on noncovalent interactions between molecules, Chemico-Biological Interactions 117 (3) (1999) 191–217.  doi:10.1016/s0009-2797(98)00097-0.
  * [48] G. Piszczek, J. C. Lee, N. Tjandra, C.-R. Lee, Y.-J. Seok, R. L. Levine, A. Peterkofsky, Deuteration of Escherichia Coli enzyme I(Ntr) alters its stability, Archives of Biochemistry and Biophysics 507 (2) (2011) 332–342.  doi:10.1016/j.abb.2010.12.022.
  * [49] J. White, V. Laux, N. Fraser, D. HeB, M. Haertlein, V. Forsyth, et al., Deuterium effects on human serum albumin in solution, Physica B: Condensed Matter 551 (2018) 208–211.  doi:10.1016/j.physb.2018.01.065.
  * [50] P. J. Nichols, I. Falconer, A. Griffin, C. Mant, R. Hodges, C. J. McKnight, B. Vögeli, L. Vugmeyster, Deuteration of nonexchangeable protons on proteins affects their thermal stability, side-chain dynamics, and hydrophobicity, Protein Science 29 (7) (2020) 1641–1654.  doi:10.1021/bi00883a002.
  * [51] A. Hattori, H. L. Crespi, J. J. Katz, Effect of side-chain deuteration on protein stability, Biochemistry 4 (7) (1965) 1213–1225.  doi:10.1021/bi00883a002.
  * [52] J. Hermans Jr, S. HA, The thermally induced configurational change of ribonuclease in water and deuterium, Biochimica et Biophysica Acta 36 (1959) 534–535.  doi:10.1016/0006-3002(59)90197-0.
  * [53] L. Sportelli, A. Desideri, A. Campaniello, Isotopic effect on the kinetic of thermal denaturation of ceruloplasmin, Zeitschrift für Naturforschung C 40 (7-8) (1985) 551–554.  doi:10.1515/znc-1985-7-817.
  * [54] G. I. Makhatadze, G. M. Clore, A. M. Gronenborn, Solvent isotope effect and protein stability, Nature Structural Biology 2 (10) (1995) 852–855.  doi:10.1038/nsb1095-852.
  * [55] R. Guzzi, L. Sportelli, C. La Rosa, D. Milardi, D. Grasso, Solvent isotope effects on azurin thermal unfolding, The Journal of Physical Chemistry B 102 (6) (1998) 1021–1028.  doi:10.1021/jp9724380.
  * [56] M. Calvin, J. Hermans Jr, H. A. Scheraga, Effect of deuterium on the strength of hydrogen bonds, Journal of the American Chemical Society 81 (19) (1959) 5048–5050.  doi:10.1021/ja01528a009.
  * [57] D. Li, W. Takarada, M. Ashizawa, T. Yamamoto, H. Matsumoto, Effect of hydrogen–deuterium exchange in amide linkages on properties of electrospun polyamide nanofibers, Polymer 229 (2021) 123994.  doi:10.1016/j.polymer.2021.123994.
  * [58] V. Nigro, R. Angelini, M. Bertoldo, F. Bruni, M. A. Ricci, B. Ruzicka, Dynamical behavior of microgels of interpenetrated polymer networks, Soft Matter 13 (30) (2017) 5185–5193.  doi:10.1039/C7SM00739F.
  * [59] I. Berndt, J. S. Pedersen, W. Richtering, Temperature-sensitive Core–Shell microgel particles with dense shell, Angewandte Chemie 118 (11) (2006) 1769–1773.  doi:10.1002/anie.200503888.
  * [60] B.-J. Niebuur, L. Chiappisi, X. Zhang, F. Jung, A. Schulte, C. M. Papadakis, Formation and growth of mesoglobules in aqueous poly(_N_ -isopropylacrylamide) solutions revealed with kinetic small-angle neutron scattering and fast pressure jumps, ACS Macro Letters 7 (10) (2018) 1155–1160.  doi:10.1021/acsmacrolett.8b00605.
  * [61] B.-J. Niebuur, L. Chiappisi, F. Jung, X. Zhang, A. Schulte, C. M. Papadakis, Kinetics of mesoglobule formation and growth in aqueous poly(_N_ -isopropylacrylamide) solutions: Pressure jumps at low and at high pressure, Macromolecules 52 (17) (2019) 6416–6427.  doi:10.1021/acs.macromol.9b00937.
  * [62] S. Sbeih, P. S. Mohanty, M. R. Morrow, A. Yethiraj, Structural parameters of soft PNIPAM microgel particles as a function of crosslink density, Journal of Colloid and Interface Science 552 (2019) 781–793.  doi:10.1016/j.jcis.2019.05.047.
  * [63] M. Brugnoni, A. C. Nickel, L. C. Kröger, A. Scotti, A. Pich, K. Leonhard, W. Richtering, Synthesis and structure of deuterated ultra-low cross-linked poly(_N_ -isopropylacrylamide) microgels, Polymer Chemistry 10 (19) (2019) 2397–2405.  doi:10.1039/C8PY01699B.
  * [64] H. M. Crowther, B. R. Saunders, S. J. Mears, T. Cosgrove, B. Vincent, S. M. King, G.-E. Yu, Poly(NIPAM) microgel particle de-swelling: a light scattering and small-angle neutron scattering study, Colloids and Surfaces A: Physicochemical and Engineering Aspects 152 (3) (1999) 327–333.  doi:10.1016/S0927-7757(98)00875-9.
  * [65] V. Nigro, R. Angelini, M. Bertoldo, B. Ruzicka, Swelling of responsive-microgels: experiments versus models, Colloids and Surfaces A: Physicochemical and Engineering Aspects 532 (2017) 389–396.  doi:10.1016/j.colsurfa.2017.04.059.
  * [66] V. Nigro, R. Angelini, M. Bertoldo, E. Buratti, S. Franco, B. Ruzicka, Chemical-physical behaviour of microgels made of interpenetrating polymer networks of PNIPAM and Poly(acrylic acid), Polymers 13 (9) (2021) 1353.  doi:10.3390/polym13091353.
  * [67] X. Wang, C. Wu, Light-scattering study of coil-to-globule transition of a poly(_N_ -isopropylacrylamide) chain in deuterated water, Macromolecules 32 (13) (1999) 4299–4301.  doi:10.1021/ma9902450.
  * [68] G. Schirò, Y. Fichou, F.-X. Gallat, K. Wood, F. Gabel, M. Moulin, M. Härtlein, M. Heyden, J.-P. Colletier, A. Orecchini, et al., Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins, Nature Communications 6 (1) (2015) 1–8.  doi:10.1038/ncomms7490.
  * [69] M.-C. Bellissent-Funel, A. Hassanali, M. Havenith, R. Henchman, P. Pohl, F. Sterpone, D. Van Der Spoel, Y. Xu, A. E. Garcia, Water determines the structure and dynamics of proteins, Chemical Reviews 116 (13) (2016) 7673–7697.  doi:10.1021/acs.chemrev.5b00664.
  * [70] P. Gallo, J. Bachler, L. E. Bove, R. Böhmer, G. Camisasca, L. E. Coronas, H. R. Corti, I. de Almeida Ribeiro, M. de Koning, G. Franzese, et al., Advances in the study of supercooled water, The European Physical Journal E 44 (11) (2021) 1–36.  doi:10.1140/epje/s10189-021-00139-1.
  * [71] A. Kyritsis, A. Panagopoulou, P. Pissis, R. S. I. Serra, J. G. Ribelles, N. Shinyashiki, Water and protein dynamics in protein-water mixtures over wide range of composition, IEEE Transactions on Dielectrics and Electrical Insulation 19 (4) (2012) 1239–1246.  doi:10.1109/ISE.2011.6084976.