Polymer 211 (2020) 123146

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Polymer
journal homepage: http://www.elsevier.com/locate/polymer

LCST polymers: Thermoresponsive nanostructured assemblies

towards bioapplications☆
George Pasparakis, Constantinos Tsitsilianis *
Department of Chemical Engineering, University of Patras, 26500, Patras, Greece

A R T I C L E I N F O A B S T R A C T

Keywords: Thermoresponsive polymers constitute an important class of materials for biomedical application due to their
LCST thermally reversible coil-to-globule transition which can be exploited in a multitude of biomedical applications
Thermoresponsive polymer spanning from triggered drug delivery systems in the form of bulk hydrogels and nanoparticles, “smart” cell
Assembly
culture setups, sensors and actuators, and separation technologies. In this perspective article we present the basic
Bioapplication
physicochemical properties of poly (N-isopropylacrylamide) (PNIPAM), which is the most widely studied ther­
moresponsive polymer with a lower critical solution temperature. We describe the basic thermodynamic and
physicochemical parameters that affect the LCST and present selective applications that utilize thermoresponsive
polymers in the form of nano-assemblies including micelles, polymersomes, microcapsules and microgels, as well
as injectable hydrogels for biomedical applications.

1. Introduction that exhibit large thermally-controlled volume changes, thermores­

Polymeric materials find numerous applications in the biomedical
field as biomaterials, fillers, structural components, sensors and actua­
tors, tissue engineering matrices and as drug delivery systems. A sub-
class of polymers for biomedical applications, the so called “smart”
polymers, can change their physicochemical properties in response to a
stimulus such as application of a pH gradient, the presence of a specific
analyte, irradiation, or heat, enabling their use as dynamic modulators
of biochemical cues at the interface of biological and biomedical sci­
ences [1–3]. Thermoresponsive polymers constitute an interesting class
of materials that can change their physicochemical properties by
application of a thermal stimulus within a narrow temperature window.
The field of thermoresponsive polymers has grown exponentially over
the last 50 years with important contributions in the fields of controlled
drug delivery, cell culture technologies and tissue engineering, separa­
tion analytical biosciences, as well as in sensors and actuators technol­
ogies [4–8]. The field was sparked by the seminal work of Heskins and
Guillet who first reported the exact thermal transition mechanism of
poly (N-isopropylacrylamide) (PNIPAM) [9], the first and most studied
synthetic thermoresponsive polymer. Soon after, landmark works fol­
lowed conceptualizing PNIPAM-based thermoresponsive hydrogels [10]
ponsive micelles that release anticancer drugs in an on-demand manner,
“smart” PNIPAM-coated surfaces for cell-sheet engineering applications
[11,12], and PNIPAM-protein conjugates with thermally-controlled
bioactivity [13–15]. The advent of reversible-deactivation radical
polymerization (RDRP) routes [16,17] and conjugation protocols via
“click” chemistries [18,19] further boosted the field as it enabled for
robust control on the macromolecular architecture on surfaces and in­
terfaces as well as the precise attachment on biomolecules and nano­
particles which was previously not possible [20].
The possibility to combine PNIPAM with additional stimuli cues
further expanded the potential uses for biomedical applications
requiring even subtler polymer multi-stimuli responses in biological
milieu even at isothermal conditions [21,22]. In this perspective article
we describe the key properties of PNIPAM and other LCST polymers, we
describe the thermodynamics of LCST and the factors that influence it,
and finally we present characteristic example applications utilizing
mainly PNIPAM as a responsive macromolecular element. Although
there are several review articles summarizing the concepts of responsive
polymers and their applications from various viewpoints, We regard the
present manuscript not as a classical review but as an introductory
reference describing key concepts of LCST-polymers, mostly in a

☆
This perspective article has been submitted for consideration in the special Issue of Polymer entitled: From “Makromolekel” to POLYMER: A Centennial Cele­
bration of Staudinger’s “On Polymerization”
* Corresponding author.
E-mail address: ct@chemeng.upatras.gr (C. Tsitsilianis).

https://doi.org/10.1016/j.polymer.2020.123146
Received 13 July 2020; Received in revised form 8 September 2020; Accepted 13 October 2020
Available online 17 October 2020
0032-3861/© 2020 Published by Elsevier Ltd.
G. Pasparakis and C. Tsitsilianis
Scheme 1. Schematic showing the coil-to-globule transition with well polymer solvation below the LCST and domination of the hydrophobic interactions above the
LCST in a), binodal diagrams for U/LCST polymers depending on polymer fraction ϕ in b) and a typical diagram of transmittance vs. temperature of an aqueous
PNIPAM sample with full transmittance below the LCST followed by complete suppression above the LCST in c).
nano-assembled form, combined with selective recent applications
related to the biomedical field in a wider context.
1.1. Thermodynamics of LCST
Polymer solvent interaction roots back to the seminal work by Flory
[23] and Huggins [24] who first described the polymer-solvent inter­
action parameter and explained the deviation of polymer solubility from
classic solubilization of small molecules, followed by the concept of free
volume [25] which was later used to explain the lower/upper critical
solution temperature phenomenon. Generally, thermoresponsive poly­
mers are categorized in two categories depending on whether they
exhibit a lower critical solution temperature (LCST) or an upper critical
solution temperature (UCST) (Scheme 1a) [26,27]. Both classes
comprise polymers that change their physicochemical properties above
a certain temperature. UCST polymers are non-soluble at low tempera­
tures and become soluble above the UCST by an enthalpic-driven pro­
cess [28]. Conversely, LCST-type polymers interact well with their
solvent at low temperatures but undergo sharp coil-to-globule transition
above their LCST and drop out of solution by an entropy increase. Both
mechanisms are rooted to the so-called hydrophobic effect [29] of water
and can be described as binary systems composed of the solvent (or a
mixture of solvents) and the polymer, with their mixability being
dependent by temperature and the polymer fraction. The phase
boundary is denoted as the binodal and corresponds to the temperature
where de-mixing occurs at each corresponding volume fraction of the
polymer (Scheme 1b) [30].
In the context of biomedical applications, by far the most studied
polymers are those interacting with water and in particular polymers
that exhibit LCST. For UCST-type polymers the reader can refer to
excellent review articles here [31,32].
As previously mentioned, PNIPAM is the most studied thermores­
ponsive polymer mainly because it has an LCST onset close to physio­
logical temperature (32 ◦ C) and it resembles the structure of isoleucine
and therefore can be regarded as a polypeptide mimic [33]. PNIPAM
undergoes an entropic coil-to-globule transition in aqueous media
(Scheme 1a) [34]. The thermally controlled mixing/de-mixing transi­
tion can be understood by the Gibbs free energy equation ΔG = ΔH-TΔS
[35]; at low temperatures (Τ<LCST) the enthalpy of mixing is negative
due to the formation of hydrogen bonds between water molecules and
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Polymer 211 (2020) 123146
the amide segments conveying a more ordered state of negative ΔS. It is
the enthalpic term at this phase that drives mixing as ΔGmix must be
negative. An increase of the temperature triggers perturbation and ul­
timately disruption of the hydrogen bonds at a temperature near the
LCST. At T > LCST, endothermic breaking of hydrogen bonding makes
the enthalpic term less negative and the entropy gain renders the
entropic term ΤΔS predominant leading to demixing and phase separa­
tion. At T > LCST, the hydrophobic interactions of the backbone and the
isopropyl segments dominate and induce polymer globule formation
which at higher concentrations can be observed as flocculation and
precipitation in aqueous media. Since the entropic coil-to-globule
transition affects mostly the re-orientation of water molecules during
de-mixing, the transitions tend to be quite faster compared to UCST
polymers which partly explains why LCST polymers are more widely
exploited in the biomedical field compared to their UCST counterparts.
It should be noted that PNIPAM in its collapsed state is not fully de-
hydrated, hydrophobic interactions are dominant above the LCST
however the globules still contain a substantial amount of water
(Scheme 1) [36–38]. The thermal transition process is fully reversible
with minor hysteresis taking place within a few microseconds upon
application of a temperature jump above the LCST [39] depending on
the polymer concentration the rate of heating/cooling of the experi­
mental, the polymer topology and confinement [40] and the presence of
hydrophobic moieties [41]. The LCST is the lowest temperature of the
binodal at which demixing is observed and should not be confused with
the cloud point temperature (Tcp) [42]. The latter is defined as the
temperature at which phase-separation occurs at a specific polymer
concentration which should be given and is usually observed by a
characteristic cloudiness of the solution (Scheme 1c). Turbidimetry
measurements with UV/Vis spectrometry coupled with temperature
control (i.e. a Peltier unit) are most commonly used to determine the
LCST, although differential scanning calorimetry, light scattering, and
nuclear magnetic resonance can also be used to determine the thermal
behavior of these materials [42,43].
1.2. Factors affecting the LCST
The LCST of PNIPAM can be affected by many parameters depending
on the molecular structure and the aqueous environment. The end-group
was shown to minimally affect the LCST when preparing polymers with
G. Pasparakis and C. Tsitsilianis
azo-initiators such as the commonly used azoisobutyronitrile (AIBN)
[44]. Minor influence of the LCST was also observed with the use of
potassium persulfate initiators which was significant only at lower
molecular weight polymer samples (ca. 2 kDa) [45]. Expectedly, soluble
end-groups such as -OH, -NH2 tend to increase the LCST while more
hydrophobic end-groups seem to decrease the LCST [44,46]. These ef­
fects have been observed by controlled polymerization routes including
atom transfer radical polymerization (ATRP) and reversible
addition-fragmentation chain transfer (RAFT) polymerizations and
generally are more pronounced at samples below ca. 50 kDa [44,47].
Polymer tacticity seems to affect the solubility as isotactic and syndio­
tactic PNIPAM are insoluble in water; insertion of isotactic blocks across
a random PNIPAM polymer chain was found to suppress the cloud point
[48,49]; ultimately, the monomer sequence [50] across the polymer
chain seems to play a critical role on the physicochemical properties in
responsive polymers and still poses a synthetic challenge that needs
further development.
The most effective way to control the exact LCST onset is the use of a
comonomer. Water soluble co-monomers contribute to the energy
required to disrupt H-bonding, hence shifting the LCST to higher tem­
peratures, while hydrophobic co-monomers tend to have a decremental
effect. Careful selection of the co-monomers should be considered with
similar reactivity ratios during the polymerization reactions in order to
achieve homogeneous incorporation of the co-monomer across the
PNIPAM polymer chain and finely control the LCST shifting with
adequate molecular fidelity. A classic example of co-monomer feed
include the use of ionizable monomers such as methacrylic acid [51],
and diethylaminoethyl methacrylate [52,53]. Since the solubility of
these co monomers can be tuned by their respective pKa, it is possible to
precisely adjust the hydrophilic-hydrophobic balance of the final
copolymer to exhibit coil-to-globule transition isothermally. This is
particularly useful for applications in vivo where large differences in the
pH of different tissues and intracellular compartments can be harnessed
to elicit triggered molecular cargo release (i.e. drugs, nucleic acids, etc.)
in a pH-dependent manner. This approach greatly expand the scope of
PNIPAM as LCST can be used as an optical readout mechanism [54], as a
reporter of ligand-receptor interactions and biomolecular events [55], as
well as hide-and-reveal [56] strategies by simple rational copolymer
design principles.
Scheme 2. Alterative to PNIPAM polymers with LCST in aqueous media.
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Polymer 211 (2020) 123146
Interestingly the interplay of confined polymer topology in combi­
nation with co-monomers that elicit co-operative interactions with
NIPAM monomer (i.e. via intramolecular H-bonding) can often lead to
unexpected manipulation motifs of the LCST as elegantly shown in a
series of studies by Richtering and co-workers [57–59].
The LCST is greatly affected by the presence of salts in aqueous
media, the so called Hofmeister salt series which are categorized as
cosmotropes and chaotropes, salts that either “make” (stabilize) or
“break” (de-stabilize) the structure of water molecules and were origi­
nally ranked according to their capacity to “salt-out” (precipitate) pro­
teins [60]. Cosmotropic salt addition leads to polarization of the water
molecules adjacent to the amide units and increase the surface tension of
the hydrophobic segments resulting in suppression of the LCST. Chaot­
ropes seem to effect on the surface tension by direct ion binding to the
amide moieties and removal of the hydration layer of the hydrophobic
isopropyl and backbone segments, resulting in salting-in of the polymer
[61].
Finally, another factor affecting the LCST, albeit less relevant to
bioapplications, is the so called cononsolvency effect [62] (usually the
use of a mixture of water with a good organic solvent such as water/­
MeOH) which tends to decrease and increase the LCST onset at moderate
and high co-solvent fractions respectively [63,64].
2. Other LCST polymers
Many polymers with H-bonding sites have a LCST (Scheme 2);
notable polymers of interest in the biomedical field include poly ((2-
dimethylamino)ethyl methacrylate)) (PDMAEMA) (pH-dependent LCST
range from 32oC to 53 ◦ C) [65,66], some poly (ethylene oxide)s (PEOs,
LCST range from 100oC to 150 ◦ C) [67,68] and poly (propylene oxide)s
(PPO, LCST 10oC–45 ◦ C) [69] within a certain molecular weight range,
hydroxypropyl cellulose (LCST, 45 ◦ C) [70], poly (N-vinyl caprolactam)
(PNVCL) (LCST at 32oC–34 ◦ C) [71], poly (ethylene glycol methacry­
late)s (LCST range from ca. 10–100 ◦ C) [72] and poly (2-oxazoline)s
(pOxs, LCST range from ca. 25–100 ◦ C) [73].
PDMAEMA has pH dependent LCST due to the ionizable tertiary
amine moiety which however raises toxicity issues for in vivo applica­
tions [74,75]. PEOs and PPOs have both been mostly exploited as tri­
block copolymers with the structure PEO-PPO-PEO which are
G. Pasparakis and C. Tsitsilianis
Fig. 1. Schematic representation of various topologies bearing LCST polymeric blocks utilized to fabricate thermoresponsive assemblies: a) A-b-B, b) (A-co-B)-b-(C-
co-D), c) B-b-A-b-B, d) A-b-B-b-C, e) A-b-(C-co-D), f) (C-co-D)-b-A-b-(C-co-D), g) protein-g-A conjugate, h) A-g-B, i) A-g-B-co-C, d) An [B-b-(C-g-D)]n (bold denotes
LCST blocks).
commercially available as Poloxamers, and Pluronics [76] and find uses
as pharmaceutical excipients. (PNVCL) in an interesting polymer as it
shows similar thermal properties with PNIPAM, and was relatively
recently polymerized by controlled polymerization methods (see for
example [77]), and also it does not degrade under in vivo conditionς
which is appealing for certain in vivo applications.
PEGMAs have been proposed as PNIPAM alternatives as they
resemble the structure of poly (ethylene glycol) which is currently
considered as the gold standard biocompatible polymer, and they endow
chemical versatility owing to their polymerization via controlled poly­
merization routes [78]. One of their drawbacks is that they can coor­
dinate heavy metal ions which could pose certain limitations for their
use. pOxs have recently emerged as a promising PNIPAM alternative,
they are synthesized by living cationic ring opening polymerization,
they do not suffer hysteresis issues during recovery (i.e. from heating to
cooling) and more importantly they exhibit protein repulsion properties
similar to poly (ethylene glycol) (PEG) which renders them attractive for
the biomedical field [79].
Elastin-like-peptides (ELPs) constitute an interesting alternative to
responsive polymers that exhibit unique physicochemical properties that
could render them viable candidates as responsive nanocarriers of
translational potential. ELPs are oligo-/poly-amino acid sequences with
the general repeating unit [aPGbG]n [80] where a = I or V, b is any amino
acid except P, and n is the number of repeating units [81–83]. ELPs un­
dergo an inverse temperature responsive transition (Tt [84], unlike the
LCST in thermoresponsive polymers) where they exist in a fully soluble
state below Tt and collapse in the form of aggregates/coacervates above
the Tt. Phase separation is a reversible phenomenon and the precise Tt can
be tuned by varying n, a, and b, and is also dependent on the ELP con­
centration and the ionic strength of the medium.
In a seminal research article, Rees et al. [85] demonstrated that a
single repeating octapeptide unit exhibited unique entropy-driven ther­
mally induced transitions in a fully reversible manner similar to its
polymeric analogue. This striking finding implies that the Tt is not a
collective phenomenon derived by the cooperative interaction between
the repeating units, but that each separate peptide unit exhibits indi­
vidual thermoresponsive properties; also, it was shown that the config­
urational entropy (ΔS) for the formation of type-II turns is independent of
the peptide sequence length. Hathorne and Bermudez [86], and Nuhn
and Klok [87] confirmed these findings by the systematic analysis of the
conformational properties of short ELPs (n = 1–8) as a function of tem­
perature changes around Tt by circular dichroism and molecular
modelling. Further studies showed that ELPs constitute excellent build­
ing blocks to form T-responsive micelles [88,89] by simple coupling with
hydrophilic polypeptide chains or water soluble polymers (i.e. PEG) with
applications in precision oncology [90] and hyperthermia therapeutics
[82].
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Polymer 211 (2020) 123146
3. LCST-based segmented macromolecules
Stimuli responsive polymeric systems have attracted tremendous
attention due to a vast variety of potential applications. Temperature is
one of the stimuli that can be used in the field of biomedicine as the
human body is thermo-regulated at about 37 ◦ C, under physiological
conditions. Moreover, hyperthermia can be applied to solid tumors and
can synergistically enhance tumor cytotoxicity when combined with
other treatments; hyperthermia preferentially increases cell membrane
fluidity as well as the permeability of tumor vasculature, facilitating the
delivery of drugs. The potential to harness the benefits of locally applied
hyperthermia motivated the design of associative macromolecules with
thermo-responsiveness in the form of block copolymers. In recent years,
the enormous growth of Macromolecular Engineering through the
development of controlled/living polymerization methods [91], has
offered numerous possibilities in designing tailor-made thermo-res­
ponsive block copolymers of various topologies (Fig. 1). As it is
well-known, block copolymers self-assemble in selective media through.
Distinct interactions, forming nanostructured assemblies of various
morphologies, i.e. micellar assemblies and/or 3D networks, depending
on their macromolecular Architecture (Fig. 1). Hence, the integration of
blocks exhibiting LCST type of behavior, endow these self-assemblies
with reversible thermo-responsiveness. For instance, in block co­
polymers/aqueous media systems, the LCST-type block can be trans­
formed from hydrophilic to hydrophobic upon heating as passing
through its LCST, inducing reversible hydrophobic association. This
kind of effect has inspired the design of “smart” thermo-responsive
polymeric and/or polymer/inorganic hybrid nanostructured assem­
blies comprising micelles, nanogels micro/nano-capsules as well as 3D
networks, targeting biomedical applications i.e. as drug delivery systems
(DDS). Bellow we demonstrate selective examples of these kinds of
thermo-responsive systems.
4. Nanostructured assemblies
4.1. Micelles
The simplest thermoresponsive block copolymer is of the A-b-B
diblock topology where a LCST-type A block is joined together with
another B block by their end-groups by a single covalent bond. Two
kinds of micelles can be formed depending on the nature of the B block,
namely amphiphilic or double hydrophilic if the B block is hydrophobic,
or hydrophilic respectively. Concerning the first class, a PNIPAM-b-
PBMA (poly (butyl methacrylate)) diblock copolymer self-assembles in
aqueous media at room temperature through hydrophobic association,
forming core-shell micelles with a PBMA hydrophobic core, stabilized by
the thermosensitive PNIPAM shell [92]. These micelles were tested as
G. Pasparakis and C. Tsitsilianis
drug carriers by loading doxorubicin in their cores. By increasing tem­
perature above the PNIPAM’s LCST, the outer hydrophilic shell, that
prevents inner core interaction with biocomponents and other micelles,
can be rapidly switched to hydrophobic, allowing drug release due to
colloidal destabilization of the micelles. The dramatic
thermo-responsive on/off switching behavior of drug release correlated
with the in vitro cytotoxicity opened opportunities to construct novel
drug delivery systems in combination with localized hyperthermia. The
same group expanded their work by replacing the hydrophobic block
with the biodegradable poly (D,L-lactide) (PLA), while the LCST of
PNIPAAm was adjusted to 39.4 ◦ C, slightly above the physiological
temperature, by incorporating N,N-dimethylacrylamide moieties. The
as-designed P(NIPAM-co-DMAAm)-b-PLA copolymer was
end-functionalized by a fluorescent molecule to fabricate
self-assembling labeled thermoresponsive micelles [93]. These micelles
showed time-dependent intracellular uptake without exhibiting cyto­
toxicity. In another study [94] it was shown that the intracellular uptake
is significantly limited at a temperature below the micellar LCST at 37
◦
C, whereas the micelles were internalized into the cytoplasm above the
micellar LCST (e.g. 42 ◦ C), being dependent on polymer concentration,
time, and temperature. Importantly, no intracellular uptake of
PEG-b-PLA micelles was observed, regardless of temperature changes
showing the role of the LCST-block. The enhanced intracellular micelle
uptake was attributed to the enhanced interactions between the micelles
and cell membranes through the dehydration of corona-forming ther­
moresponsive polymer chains. This temperature dependence of the
micelle outer shell with the cell membranes was further corroborated by
our group with the use of P(PEGMA)s as the T-responsive element which
enabled the co-delivery of multiple drug combinations both in the form
of micelles [95] or polymer-coated liposomes [96] (Fig. 2).
Another PNIPAM-based copolymer with sophisticated macromolec­
ular architecture, was designed as DDS based on a tadpole-shaped,
linear-cyclic diblock copolymer, consisting of the biodegradable hy­
drophobic linear pol (ε-caprolactone) (PCL) and the thermoresponsive
macrocyclic PNIPAM [(c-PNIPAM)-b-PCL] [97]. Compared with the
linear diblock copolymer counterpart, (l-PNIPAM)-b-PCL,
Fig. 2. P(PEGMA) based thermoresponsive block copolymers synthesized by RAFT polymerization and their applications as standalone micellar and liposome
dispersions for hydrophobic and hydrophilic drug loading, respectively. LCST-driven disruption leads to augmented drug release rates as well as higher cellular
uptake due to augmented cell membrane interactions (adapted with permission from references [95,96]).
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Polymer 211 (2020) 123146
thermoresponsive micelles self-assembled from (c-PNIPAM)-b-PCL,
exhibited lower LCST and improved drug loading and releasing capac­
ity. These results indicate the importance of macromolecular architec­
ture of thermoresponsive block copolymers, on the self-assembling,
thermal phase transition properties and their functions as drug nano­
carriers for controlled release.
Double hydrophilic block copolymers constituted of PEG (hydro­
philic and biocompatible with “stealth” properties) and thermosensitive
N-(2-hydroxyethyl)methacrylamide-oligolactates, P(HEMAm-Lacn)
were designed, as efficient drug carrier systems for therapeutic appli­
cations [98]; conveniently, the LCST of the thermosensitive block can be
tuned by the number of lactate moieties thus a block copolymer PEG-b-P
(80%HEMAm-Lac2)-co-(20%HEMAm-Lac4), exhibiting low LCST,
self-assembled in room temperature forming micellar carriers with fast
degradable core and a PEG stabilized shell. Under physiological condi­
tions (pH 7.4 and 37 ◦ C), the micelles started to swell after 4 h and were
fully destabilized within 8 h due to hydrolysis of the lactate side chains.
This destabilization profile seems advantageous for in vivo use because
the observed induction period is just long enough to allow accumulation
of the micelles after intravenous administration at their site of action, e.
g., a tumor via the enhanced permeation and retention (EPR) effect [99,
100].
An interesting double hydrophilic diblock copolymer exhibiting two
LCSTs in distinct blocks were designed, based on PNVCL (Fig. 3) [101].
The first block is a statistical copolymer of VC with the hydrophobic
monomer 3-methyl-N-vinylcaprolactam P(MVC-co-VC) with LCST1
around room temperature (19–27 ◦ C). The second block is a statistical
copolymer of VC with the hydrophilic monomer N-vinylpyrrolidone
(VP) P(VC-co-VP) with adjusted LCST2 slightly above physiological
temperature (41–42 ◦ C). The diblock copolymer P(MVC-co-VC)-b-P
(VC-co-VP) self-assemble above the LCST1 forming micelles, enabling
drug encapsulation in their hydrophobic P (MVC-co-VC) cores. Upon
increasing temperature above LCST2, the dehydrated P(VC-co-VP)
corona blocks associate leading to colloidally unstable aggregates, fol­
lowed by drug release. Moreover, the elevated temperature can trigger
the loaded micelle aggregation/accumulation within the tumor
G. Pasparakis and C. Tsitsilianis Polymer 211 (2020) 123146

Fig. 3. Schematic representation of a double hydrophilic diblock with double LCST response, associating upon heating, (from ref. [102] by permission of ACS).

resulting in enhanced passive targeting.
Triple thermoresponsive triblock terpolymers, comprising three
different blocks exhibiting resolved thermal transitions of the LCST type
[poly (N-n-propylacrylamide) (LCST ~22 ◦ C), poly (methoxydiethylene
glycol acrylate) (LCST ~39 ◦ C), and poly (N-ethylacrylamide) (LCST
~73 ◦ C)] have also been synthesized and their self-assembly, by step­
wise thermal switching, was explored in respect of the block sequence
(ABC, BAC, and ACB) [103]. The self-organization (micellization) of
such terpolymers and their molecular cargo ability of hydrophobic
agents (Nile Red probe) in the collapsed lower LCST block forming the
hydrophobic core of the micelles, were found to depend strongly on the
position of the blocks across the macromolecular chain.
Besides passive targeting DDS, attempts have been undertaken to
design active targeting thermoresponsive micellar nanocarriers (Fig. 4).
Thermoresponsive (PEO-PPG-PEO (Pluronic F127, commercially avail­
able) covalently bonded with poly (d,l-lactic acid) (F127-PLA, abbrevi­
ated as FP) copolymer micelles were developed and decorated with
folate (FA) acting as the targeting functional moiety. The system
exhibited LCST at 39.2 ◦ C. Thus at 37 ◦ C, a small amount of encapsulated
anticancer drug DOX was released from the self-assembling micelles,
while at a temperature slightly above LCST (40 ◦ C), the shrinkage of
thermoresponsive PEO-PPG-PEO segments induced rapid release of DOX
and rapid increase of the drug concentration locally [104]. As demon­
strated, this copolymer has excellent cytocompatibility, and the

Fig. 4. Schematic representation of thermosensitive nanocarrier working as a targeted drug delivery system, (from ref. [107] by permission of ACS).

6
G. Pasparakis and C. Tsitsilianis
FA-decorated FP micelles exhibited improved cellular uptake and higher
cytotoxicity against folate receptor (FR)-overexpressed HeLa cells.
Importantly, the cytotoxicity of DOX-loaded FA-FP micelles against
HeLa cells was significantly more pronounced under hyperthermia (40
◦
C) compared to physiological temperature, showing promising poten­
tial as a drug nanocarrier for anticancer applications.
Non-linear segmented copolymers have also been employed as DDS.
Folate-decorated thermoresponsive micelles based on the star-shaped
amphiphilic block copolymer bearing four [poly (ε-caprolactone)-b-2s
(poly (N-isopropylacrylamide-co-acrylamide)-b-methoxy poly (ethylene
glycol)/poly (ethylene glycol)-folate)] arms (i.e., 4s [PCL-b-2s (P
(NIPAМ-co-AAm)-b-MPEG/PEG-FA)] were developed for the tumor
targeted delivery and temperature-induced controlled release of pacli­
taxel (PTX) [105]. The cytotoxicity studies showed that the transport of
PTX by these micelles into cancer cells was higher than that by the
commercial PTX formulation Tarvexol. More thermoresponsive nano­
systems based on polymeric micelles can be viewed in previous reviews
[27,106].
4.2. Polymersomes
Polymersomes constitute a special class of polymeric nanostructured
assemblies with vesicular morphology, capable to carry hydrophilic and
hydrophobic therapeutic agents in their lumen and membrane respec­
tively, rendering them promising DDSs. Thermoresponsive polymer­
somes have been designed using segmented copolymers and/or
terpolymers incorporating thermoresponsive blocks [108]. Most of these
systems are focused on heating induced polymersome formation and
some with the effect of temperature on the controlled delivery of ther­
apeutics, for example polymersomes formed by self-assembling of
methoxy-poly (ethylene oxide)-b-poly (e-caprolactone)-b-poly (N-iso­
propylacrylamide) (mPEO-b-PCL-b-PNIPAM) triblock terpolymers
[109]. These polymersomes can effectively act as a drug release carrier
of indomethacin at physiological conditions, that is, store the drug at a
lower temperature and release it at a higher temperature.
Polymersomes endowed with magneto-thermal ability (by inducing
hyperthermia), were prepared by the self-assembly of poly (isoprene)-b-
PNIPAM amphiphilic block copolymer, encapsulating monodisperse
hydrophobic superparamagnetic iron oxide nanoparticles (SPION)
within their hydrophobic compartment. A water-soluble dye, calcein,
entrapped in the lumen of the vesicles was used to evaluate this system
Fig. 5. Formation of T-responsive block copolymersomes and their polyelectrolyte crosslinking conferring additional colloidal stability (reproduced from ref. [112]
with permission).
7
Polymer 211 (2020) 123146
as a triggerable DDS. Magnetic heating of the embedded SPIONs led to
increased bilayer permeability through dehydration of the thermores­
ponsive PNIPAM block. Thus, calcein could be released in controlled
doses solely via exposure to pulses of an alternating magnetic field. This
work demonstrates controlled drug release potential applications that
combines rational polymersome design with addressed external mag­
netic field-triggered release [110].
The coexistence of thermo- and pH-responsive (weak poly­
electrolytes) blocks in the macromolecular chain endows polymers with
dual responsiveness. To this direction, multicompartmental polymer­
somes were fabricated by star-graft quarterpolymers, composed of hy­
drophobic PS and pH-sensitive P2VP-b-PAA arms, emanated from a
common nodule, enriched by thermo-sensitive PNIPAM grafts, cova­
lently bonded on the PAA block-arms. These pH/thermo-sensitive pol­
ymersomes were evaluated as nanocarriers for co-delivery of
doxorubicin (hydrophilic) and paclitaxel (hydrophobic) anticancer
chemotherapeutic agents, encapsulated in different compartments (i.e.
lumen and hydrophobic membrane, respectively). The loaded poly­
mersomes were found to be internalized efficiently in human lung
adenocarcinoma epithelial cells. Enhanced release was observed for
both payloads at pH 6.0 and physiological temperature (37 ◦ C). At the
same total drug level, co-delivery of these drugs with the polymersomes
had enhanced cytotoxicity and induced significantly higher cell
apoptosis in the cancer cell line, compared to the polymersomes loaded
with each of the two drugs separately [111].
The combination of T-responsive polymers with polyelectrolytes
constitutes a powerful approach as it harnesses strong Coulombic in­
teractions and the thermally driven transition properties; for example,
McCormick et al. [112] coupled PNIPAM with poly (N-(3-aminopropyl)
methacrylamide P (AMPA) which was crosslinked with poly (sodium
2-acrylamido-2-meth-ylpropanesulfonate) (PAMPS) via interpolyelec­
trolyte interactions to “lock” polymersomes (Fig. 5.); the same group
further extended their approach to crosslink PNIPAM-rich block copol­
ymer micelles [113].
In a series of studies, Plamper et al. [114–116] explored the combi­
nation of LCST triggering as a means to kinetically control the shape of
polyelectrolyte nanoassemblies and their compartmentalization; this
approach opens up the opportunity to develop multi-stimuli responsive
nanocontainers capable to carry and deliver multiple molecular cargos
of different solubility. In a similar context Sumerlin et al. [117] proposed
the polymerization-induced thermal self-assembly (a variation of the so
G. Pasparakis and C. Tsitsilianis
called polymerization induced self-assembly PISA [118]) where the
formation of nano-objects (i.e. spherical or worm-like nanoassemblies)
with tunable shape can be finely controlled via in situ control of the
degree of polymerization and the application of thermal stimulation that
modulates the packing parameter in aqueous media.
PNIPAM conjugated to proteins (BSA-NH2) to obtain thermally
responsive microcompartments called “proteinsomes”, by analogy to
polymersomes, have also been reported, expanding the design capabil­
ities towards thermoresponsive biological hybrid vesicular systems. By
grafting PNIPAM chains onto the protein surface, temperature-
dependent chain conformational transition can be exploited to pro­
duce stimulus-responsive proteinosomes that exhibit self-mediated
gated permeability at water/water phase boundaries. In principle, this
offers the ability not only to produce targeted microcontainers but also
to use localized cooling of targeted cells and tissues for the temperature-
mediated release of bioactive payloads [119].
4.3. Microcapsules
Polymeric vesicular carriers, namely, microcapsules and/or nano­
capsules have emerged for potential applications in biomedicine. Among
them, systems incorporating LCST type thermoresponsive polymers
have been designed and prepared. An interesting example is the fabri­
cation of ultrathin thermoresponsive microcapsules through direct co­
valent layer-by-layer (LbL) assembly by “click” chemistry using azido-
and acetylene-functionalized poly (N-isopropylacrylamide) (PNIPAM)
“clickable” random copolymers [120]. The synthesized poly [N-iso­
propylacrylamide-co-(trimethylsilyl)propargylacrylamide] (PNIPA­
M-Ace) and poly (N-isoropylacrylamide-co-3-azideopropylacrylamide)
(PNIPAM-Az) after removing the protective trimethylsilyl groups, were
assembled alternately onto azido-modified silica particles in aqueous
media through “click” reactions. After removing the silica template, the
microcapsules remained stable because of the presence of the covalently
bonded triazole units (Fig. 6).
The microcapsules exhibited swelling/deswelling behavior by
responding reversibly to temperature changes of the medium. Evalua­
tion study on the permeability of microcapsules revealed that the mi­
crocapsules with tighter packing wall are selectively permeable to
molecules and show potential for applications for the encapsulation of a
variety of materials.
Dual pH/thermo responsive microcapsules fabricated by the LbL
technique through non-covalent interactions have also been designed.
Fig. 6. Schematic Representation of the Preparation of Covalently Stabilized Thermoresponsive Microcapsules Through Layer-by-Layer Assembly Using Click
Chemistry, (from ref. [120] by permission of ACS).
8
Polymer 211 (2020) 123146
Particularly, star-graft quarterpolymers (SGQP) PSn [P2VP-b-(PAA-g-
PNIPAM)]n (PS: polystyrene, P2VP: poly (2-vinylpyridine), PAA: poly
(acrylic acid)) containing two kinds of arms, one is a shorter hydro­
phobic PS arm, the other is a pH responsive hydrophilic P2VP-b-PAA
block copolymer arm with grafted PNIPAM chains on the outer PAA
block were assembled with tannic acid through H-bonding on a sacri­
ficial silica microparticle, forming multilayered nanostructured micro­
capsules. Due to the presence of weak polyelectrolytes and PNIPAM in
the layers, this microcapsule exhibited pH and thermosensitivity. It was
shown that both hydrophobic (small molecules) and hydrophilic (mac­
romolecules) cargos can be encapsulated in the shell or the lumen of the
microcapsules, concurrently. A programmable and sequential release of
hydrophobic and hydrophilic molecules can be triggered independently
by temperature and pH variations. These stimuli affect the hydropho­
bicity and chain conformation of the star block copolymers to initiate
out-of-shell release (elevated temperature), or change the overall star
conformation and interlayer interactions to trigger increased perme­
ability of the shell and out-of-core release (pH). Reversing of the stim­
ulus order, completely alters the release process allowing pre-
programmed, on-demand, release opposite sequences without mutual
interference [121].
A different strategy to fabricate pH/thermo responsive organic/
inorganic hybrid microcapsules, enabling multiple loading and pro­
grammable release was reported recently. PNIPAM surface modified
stellate mesoporous silica nanoparticles were used as nanocarriers for
one guest compound (e.g. Nile red), serving simultaneously as Pickering
emulsifiers to stabilize oil-in-water droplets. These droplets contain pH-
responsive monomers and another guest molecule, 5 (6)-carboxy­
fluorescein diacetate (CFDA). Upon UV irradiation polymerization, the
pH-responsive monomers were converted into polymer microcapsules.
The release of both cargos could be selectively activated by changing the
temperature or the pH value [122].
Partially biodegradable thermoresponsive self-folding capsules,
capable of controlled capture and release of cells (e.g. yeast cells) in
response to a temperature have been demonstrated by using star-like
patterned polycaprolactone and PNIPAM crosslinked bilayers. Backing
yeast cells were deposited on the patterned polymer bilayer from buffer
dispersion at elevated temperature when PNIPAM is in the collapsed
state. Upon decreasing temperature, due to PNIPAM swelling, folding of
the polymer capsules occurs, encapsulating the cells which can be
delivered upon heating, inducing deswelling of PNIPAM and unfolding
of the capsule (Fig. 7) [123].
G. Pasparakis and C. Tsitsilianis
Fig. 7. Schematic and bright field optical microscopy images of the folding star-shaped polymer bilayer. Swelling of the thermoresponsive hydrogel layer at lower
temperature increases stress in the film that results in bending of the star arms and folding (from ref. [123] by permission of RSC).
4.4. Microgels
T-responsive gels harness the large volume changes driven by tem­
perature stimuli at the micro- or nano-scale finding interesting appli­
cations mostly as triggerable molecular cargo release systems [124]. Of
particular interest for bioapplications are micro-/nano-sized PNIPAM
gels owing to their relative ease of fabrication, their inherent capability
to be combined with different co-monomers and as recently reported
their excellent protein repellent properties [125] which render them
suitable drug carriers for in vivo delivery applications.
Initial reports on PNIPAM microgels were based on surfactant-free
free radical polymerization in presence of crosslinkers with the use of
persulfate initiators at 60–70 ◦ C. This protocol is sometimes referred to
as “precipitation polymerization” as the high temperature induces
instant collapsing of NIPAM oligomers during the propagation step and
produces well dispersed microgel particles [126]. Variations of this
protocol include the incorporation of surfactants such as sodium dodecyl
sulfate, or the addition of macro-stabilizers in the form of macro­
monomers or macro initiators in the case of CRP methodologies. Pre­
cipitation polymerization is somewhat advantageous to classic emulsion
polymerization as only a single aqueous reaction phase is needed which
simplifies reaction setups and purification steps. Lyon et al. [127] fully
exploited the robustness of precipitation polymerization to construct
core-shell microgel particles with fully detached compartments; first
they grew seed PNIPAM particles with the use of N,N′ -methylenebis
(acrylamide) (BIS) (Fig. 8) followed by the growth of a sacrificial shell
by replacing BIS with N,N′ -(1,2-dihydroxyethylene)bisacrylamide. A
final precipitation polymerization step by using BIS again as crosslinker
leads to the formation of a final outer shell. Treatment of the particles
with sodium periodate results in hydrolysis of the middle sacrificial shell
affording detached core-shell T-responsive microgel particles.
For more complex molecular architectures, emulsion polymerization
remains the method of choice, especially, for protocols requiring mul­
tiple monomers [128,129]. In addition, core-shell microgels can be
obtained by the sequential addition of a second monomer either in situ
or by using an additional seed-polymerization step. In recent years, the
fabrication of microgel particles with the use of microfluidic devices
have also been employed as they allow for superior homogeneity and
control on the microscale architecture and more importantly, they can
9
Polymer 211 (2020) 123146
integrate living cell populations during fabrication which enables the in
situ generation of cell encapsulating microparticles in the form of par­
ticle dispersions, or as injectable soft biomaterials [130,131].
Generally, T-responsive microgels retain their temperature respon­
sive properties and in analogy with bulk hydrogels exhibit large changes
of their volume albeit in a more confined space and hence the transition
temperature is often referred to as volume phase transition temperature
(VPTT). In addition to cell delivery systems, they have found numerous
applications as stimuli responsive drug delivery systems in precision
medicine, as T-recoverable catalysts for homogeneous and heterogenous
catalysis applications, fluorescent reporters and as building blocks for
photonic crystals with T-switchable optical properties [132,133].
Microgels’ confined topology allows for facile integration with micro­
fluidic devices [134] owing to their large surface to volume areas which,
in addition to their T-induced volume changes, can be exploited for
efficient surface modification of electrodes and further loading of en­
zymes [135,136] for biosensing applications that exploit and also their
T-induced volume changes of the microgels as readout mechanisms with
very low detection limitscan be exploited for efficient surface modifi­
cation of electrodes and further loading of enzymes [135,136].
4.5. Injectable hydrogels
Hydrogels are three-dimensional (3D) soft materials consisting usu­
ally of a polymeric network capable to entrap high amounts of water,
making them suitable for medicinal applications as DDS and/or scaffolds
for tissue engineering. Two types of hydrogels can be distinguished,
depending on the chemical or physical crosslinking of the polymeric
gelator, leading to permanent or reversible networks respectively.
Hydrogels can be classified according to the origin of the gelator in
natural (e.g. polysaccharides), synthetic (copolymers) and semi­
synthetic (natural/synthetic combinations); by incorporating LCST
macromolecular chains in the gelator, thermoresponsive hydrogels can
be achieved.
A special category, namely injectable hydrogels, are those gels that
are formed in situ after injection of a polymeric solution of freely moving
entities (either free or associated chains) capable to form a network by
crosslinking. In the case of thermoresponsive hydrogels, two strategies
have been developed so far. 1) chemical crosslinking comprising LCST
G. Pasparakis and C. Tsitsilianis Polymer 211 (2020) 123146

Fig. 8. Core-shell PNIPAM microgels with fully detached compartments synthesized by three consecutive precipitation polymerization steps (core (C), core-shell
(CS), and core-double-shell (CDS)) followed by periodate hydrolysis of the middle shell. Under the atomic force microscope, the outer shells look almost
completely flat due to the complete absence of structural rigidity (adapted with permission from ref. [127]).

polymers bearing functions reacting in situ to form a network and 2)
physical crosslinking of block copolymer type macromolecules which
relies on the hydrophilic-to-hydrophobic transition above the LCST of
the thermosensitive blocks of the gelator, inducing self-association of
the freely moving entities into a spanning network. In the second
strategy we can distinguish two main trends. 1) use of amphiphilic co­
polymers that form micellar self-assemblies at room temperature before
injection and 2) double hydrophilic copolymers that self-assemble in the
physiological temperature after injection. In the former case immobili­
zation of the micelles occurs either by jamming or by secondary thermo-
association (Fig. 9).
The research activities in the area of thermogels is very extensive due
to potential bioapplications, namely: 1) minimally invasive drug de­
livery; 2) injectable tissue engineering (3D cell/stem cell culture); 3)
post-surgical treatments for adhesion prevention; 4) long-term magnetic
resonance imaging (MRI) contrasting; 5) transcatheterarterial emboli­
zation; 6) 3D live cell imaging for cellular processes; and 7) wound
dressings (Fig. 10) [137].
Several examples of thermoresponsive injectable hydrogels can be
viewed in previous reviews and under different contexts [10,27,
137–144]. In the following paragraphs, some new trends are briefly
demonstrated.
Polysaccharides are very attractive biocompatible and biodegrad­
able natural biopolymers, bearing functional groups susceptible to easy
modification toward designing thermo-induced injectable hydrogels
[145,146]. Grafting methodologies allow attachment of LCST polymers
as pendant chains, endowing the resulting semisynthetic graft co­
polymers with thermoresponsiveness. Efforts have been made to fine
tune the rheological properties including injectability. For instance so­
dium alginate, grafted by a thermo-responsive random copolymer of
PNIPAM, enriched with 10% mol of the hydrophobic N-tert-butylacry­
lamide, Alg-g-P(NIPAM90-co-NtBAM10) showed sol-gel transition (Tgel)
at about 32 ◦ C and excellent shear- and thermo-induced injectability
[147]. The presence of the hydrophobic moieties in the thermores­
ponsive PNIPAM grafting chains (stickers) regulate their LCST as well as
the strength of hydrophobic association, in terms of exchange dynamics
of the stickers of the network that control the rheological properties.
However, the Tgel and the gel properties are influenced by a number of
factors, namely, gelator concentration, grafting density, nature and
length of grafts as well as by the environmental conditions like

Fig. 9. Schematic representation of gelation above the LCST (red color illustrates the LCST blocks): A) jamming of micelles formed upon heating of double hy­
drophilic diblock copolymers; B) double hydrophilic triblocks self-assemble in a micellar network by bridging; C) amphiphilic triblock micelles, with LCST chains in
the corona, associate.

10
G. Pasparakis and C. Tsitsilianis
Fig. 10. Potential applications of a thermogelling material, (from ref. [137] by permission from RSC).
temperature, pH, salinity and other solutes, which all have to be
co-evaluated to target tailor-made hydrogel properties for specific
applications.
The use of PNIPAM-based random copolymers instead of the ho­
mopolymer PNIPAM can be utilized to introduce biodegradability. An
interesting example of this concept is the bioresorbable polymer
injectable hydrogels formed by poly (N-isopropylacrylamide-co-
dimethyl-c-butyrolactone acrylate-co-acrylic acid) containing 7.00 mol
% of the hydrolyzable dimethyl-c-butyrolactone acrylate monomer
[148].
Stem cell transplantation using injectable hydrogel is an application
requiring a two-step gelation before and after injection in order to
protect the cells during injection and to provide adequate cell immobi­
lization in the target tissue. Shear/thermo induced injectability fulfill
these requirements. For this purpose, a two-component system has been
developed comprising aldehyde-modified hyaluronic acid (HA-ALD)
and the thermoresponsive hydrazine-modified elastin-like protein (ELP-
HY). By mixing the polymeric components at room temperature a shear-
induced injectable hydrogel is formed through reversible dynamic co­
valent hydrazone bonds. Upon heating at 37 ◦ C a secondary physical
crosslinking occurs, due to ELP LCST type thermal phase transition (see
for example [149]), mechanically reinforcing the hydrogel (Fig. 11).
This system enables.
Cell culture for three weeks post injection. Yet, the encapsulated cells
maintain their ability to differentiate into multiple lineages, including
chondrogenic, adipogenic, and osteogenic cell types [150].
5. Conclusions and outlook
PNIPAM remains the polymer of choice for applications requiring
materials with stimuli responsive polymers due to its synthetic
11
Polymer 211 (2020) 123146
versatility and the possibility to be combined with other monomers to
confer additional isothermal responsiveness towards other stimuli such
as pH gradients, irradiation, or the presence analytes. PNVCL-based and
pOx-based polymers are also emerging especially for applications where
non-degradability is a prerequisite while PEGMAs constitute an attrac­
tive alternative owing to their resemblance of PEG and their capacity to
be easily polymerized via controlled/living polymerization reactions.
Arguably ELPs will continue to be developed and show the highest po­
tential to for direct in vivo applications owing to their degradability and
their natural polypeptide structure.
T-responsive materials have now been diffused into niche areas
beyond classic polymer chemistry and materials science and they are
invaluable laboratory tools albeit mostly for proof-of-concept applica­
tions. The field has matured enough to expect practical applications to
continue to emerge not only in the biomedical field but also in the fields
of catalysis, separation and purification technologies and biosensing
applications; indeed patent filings including the word “poly (N-iso­
propylacrylamide)” have linearly increased in the last 10–20 years and
therefore it remains to be seen whether these fascinating polymers will
be integrated in out-of-the-lab applications. Of note, there are still
concerns about the toxicity of PNIPAM [151–153] and hence certain in
vivo applications may be hampered which potentially leaves room for
other alternative LCST polymers to be used for clinical practice. More
realistic expectations should lie on in vitro and/or ex vivo applications
such as molecular diagnostics, biosensing and integration with micro­
fluidic devices.
Finally, there is a clear trend towards more refined polymer topol­
ogies and architectures owing to the rapid development of controlled
polymerization methods and conjugation protocols (i.e. “click” chem­
istries, bio-orthogonal coupling reactions, etc.); these advances have
allowed for unprecedented molecular fidelity which was not possible to
G. Pasparakis and C. Tsitsilianis Polymer 211 (2020) 123146

Fig. 11. Injectable ELP–HA hydrogels. a) ELP–HA is composed of hydrazine-modified elastin-like protein (ELP-HYD) and aldehyde-modified hyaluronic acid (HA-
ALD). b) Schematic of ELP–HA hydrogel formation. c) Photographs demonstrating the injectability and rapid self-healing of ELP–HA hydrogels, (from ref. [150] by
permission of Wiley VCH).

achieve 20 years ago (a classic example-testament of these advances is
the so-called “grafting from” approach directly from protein molecules
[154]); such synthetic precision constitutes a major synthetic milestone
enabling the potential use of LCST polymers in clinical areas requiring
pharmaceutical grade materials and good manufacturing protocols.
More robust chemistries have also lead to the easier coupling of
T-responsive polymers with additional stimuli-responsive components
(i.e. pH-responsive polymers, magnetic/plasmonic nanoparticles, etc.)
[21,155] which have already led to the synthesis of multi-stimuli
responsive materials with complex behavior and often exotic
properties, which we expect to continue to be developed in the near
future.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

12
G. Pasparakis and C. Tsitsilianis Polymer 211 (2020) 123146

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