European Polymer Journal 124 (2020) 109488

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European Polymer Journal

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Thermoresponsive behavior of poly(DEGMA)-based copolymers. NMR and T

dynamic light scattering study of aqueous solutions
⁎ ⁎,1
Rafał Konefał , Jiří Spěváček , Gabriela Mužíková, Richard Laga
Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

A R T I C LE I N FO A B S T R A C T

Keywords: We studied temperature behavior of diblock copolymers of poly[2-(2-methoxyethoxy)ethyl methacrylate]

Thermoresponsive polymer (PDEGMA) with poly(N-(2-hydroxypropyl)methacrylamide and a statistical copolymer PDEGMA-co-N-pro-
Poly(2-(2-methoxyethoxy)ethyl methacrylate) pargylmethacrylamide in aqueous solutions. NMR spectra and dynamic light scattering showed that the copo-
(PDEGMA) lymers exhibit a reversible phase transition which depends on the composition of the block copolymer and
PHPMA
slightly on polymer concentration. In contrast to statistical copolymer, different behavior of proton groups in
Aqueous solution
NMR
polymer backbone (and in the nearest side chain OCH2 group) on the one hand and in other side chain groups of
Spin-spin relaxation times T2 the PDEGMA on the other hand was observed for diblock copolymers. Additionally, two types of water molecules
NOESY were detected in solutions of the diblock copolymers above the phase transition –“free” with long and “bound”
Dynamic light scattering with short spin-spin relaxation times T2. NOESY spectra indicate that a core- to- shell morphology is unlikely in
these block copolymers. This report emphasizes the importance of understanding of the self-association of co-
polymers in solution on the molecular level and provides important information for the design of “smart”
thermoresponsive polymer-based drug delivery systems.

1. Introduction isopropylacrylamide) (PNIPAm) with LCST around 305 K [12–15].

Stimuli-responsive polymers have acquired great attention in a field
of polymer biomaterials. These polymers after small external stimuli,
such as changes in temperature, pH, ionic strength, light irradiation or
complexation with appropriate molecules, significantly alter their
physical or chemical properties [1–5]. Among the miscellaneous stimuli
aforementioned, due to non-invasive treatment and a wide range of
applications such as drug delivery, tissue engineering, bioseparation,
thermoresponsive films, oil-gas industry and nanoreactors, thermo-
responsive polymers are the most extensively investigated [6–10]. In
the field of thermoresponsive synthetic polymer biomaterials, most
studies focus on materials that exhibit phase separation in water with
the lower critical solution temperature (LCST) ranging from ~293 K to
308 K [11]. This can be very useful, for example, in terms of drug de-
livery purposes, since the polymers having LCST values in this tem-
perature range can be readily and reproducibly processed and dissolved
at room temperature, at which their chains occur in a random coil
conformation (soluble form), while their chains collapse under phy-
siological conditions (e.g., when introduced into the body) to form
compact globuli (insoluble particles form). In particular, the best
known and most widely studied polymer from this group is poly(N-
However, in recent years, polymer chemists reported many interesting
alternatives to PNIPAm [16–19]. One group of such polymers are
polymethacrylates containing short oligo(ethylene glycol) (OEG) side
chains. Generally, the phase transition temperatures of these polymers
depend namely on the length of the OEG side chain. For instance, poly
(2-(2-methoxyethoxy)ethyl methacrylate) (PDEGMA) with two ethy-
lene glycol units, and poly(2-[2-(2- methoxyethoxy)ethoxy] ethyl me-
thacrylate) (PTEGMA) with three ethylene oxide units shows LCST
around 299 K and 325 K, respectively. Polymers with longer side chains
(4–9 ethylene glycol units) exhibit phase transitions between 333 and
363 K. A further advantage of those polymers is possibility of tuning of
their LCST to the application requirements. For example, LCST values
below physiological temperatures (i.e. 310 K) can be achieved by co-
polymerizing OEGMA with various hydrophobic comonomers. More-
over, studies of the interactions of POEGMAs with a number of cell lines
denote that these polymers are non-toxic and biocompatible [20–23].
Taking into account the abovementioned facts, copolymers based on
the PDEGMA appear to be suitable candidates for biomedical applica-
tions.
In aqueous media, block copolymers where one component is hy-
drophilic and the other thermoresponsive can form various self-

⁎
Corresponding authors.
E-mail addresses: konefal@imc.cas.cz (R. Konefał), spevacek@imc.cas.cz, spevac@seznam.cz (J. Spěváček).
1
Emeritus scientist (J.S.)

https://doi.org/10.1016/j.eurpolymj.2020.109488
Received 4 November 2019; Received in revised form 2 January 2020; Accepted 6 January 2020
Available online 07 January 2020
0014-3057/ © 2020 Elsevier Ltd. All rights reserved.
R. Konefał, et al.
assembled structures (micelles, vesicles, microgels or nanoparticles) at
elevated temperature [24,25]. Examples of all those nanostructures
obtained in polymers based on different POEGMAs are listed in an ex-
cellent review recently published by Szweda et al. [20].
Among the methods used to investigate the temperature behavior of
the LCST-type polymer systems such as cloud point measurement, in-
frared (IR) spectroscopy, light scattering, small angle X-ray scattering,
calorimetry etc. [26–31], NMR spectroscopy can provide quantitative
information on the LCST phase separation behavior [32,33]. Relaxation
time and diffusion experiments measurements offer possibility to follow
the changes in molecular motions of polymer and water in solution.
Additionally, the 2D 1H–1H NOESY spectra are extremely valuable to
clarify conformational problems of macromolecules. In last decade, 1H
NMR spectroscopy, NMR relaxation times and 2D 1H–1H NOESY tech-
niques were successfully applied to study various single- and multi-
component thermoresponsive polymer systems in aqueous solutions
[32–39].
In the present work we applied 1H NMR spectroscopy, 1H spin-spin
relaxation times (temperature and time dependences) and 2D nuclear
Overhauser effect spectroscopy (NOESY) at various temperatures (ap-
plied only to block copolymer) in combination with dynamic light
scattering (DLS) to study temperature-induced phase separation in
aqueous solutions of block copolymers composed of the hydrophilic
poly(N-(2-hydroxypropyl)methacrylamide (PHPMA) block and ther-
moresponsive PDEGMA block (with different molecular weights of
PDEGMA block) and P[(DEGMA)–co-N-propargylmethacrylamide
(PGMA)] statistical copolymer. Since both PHPMA and PDEGMA are
biocompatible, non-toxic and non-immunogenic materials, those co-
polymers are suitable for application in biomedicine and/or drug and
gene delivery systems.
2. Materials and methods
2.1. Chemicals
(RS)-1-Aminopropan-2-ol; 4,4′-azobis(4-cyanovaleric acid) (ACVA);
azobisisobutyronitrile (AIBN); 4-cyano-4-(phenylcarbonothioylthio)
pentanoic acid (CTA-ACVA); 2-cyano-2-propyl benzodithioate (CTA-
AIBN); N,N'-dicyclohexylcarbodiimide (DCC); 4-dimethylaminopyr-
idine (DMAP); N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride (EDC); magnesium sulfate; methacrylic acid; methacryloyl
chloride; 2-(2-methoxyethoxy)ethyl methacrylate (DEGMA); propargy-
lamine; sodium carbonate and thiazolidine-2-thione (TT) were pur-
chased from Sigma-Aldrich, Czech Republic. All solvents used in this
work were of high-purity grade with extremely low water levels pur-
chased from VWR, Czech Republic.
2.2. Synthesis of monomer, initiator and RAFT agent
N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized by
reacting methacryloyl chloride with (RS)-1-aminopropan-2-ol in di-
chloromethane in the presence of sodium carbonate according to pro-
cedure described in [40].
N-Propargylmethacrylamide (PGMA) was synthesized by reacting
methacrylic acid with propargylamine in dichloromethane (DCM) in
the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride (EDC). A mixture of methacrylic acid (1.567 g, 18.2 mmol)
and EDC (4.525 g, 23.6 mmol) was dissolved in 86 mL of DCM and
cooled to 255 K. Propargylamine (1,0 g, 18,2 mmol) and a few crystals
of DMAP were added to the cooled solution and the reaction mixture
was stirred 2 h at 255 K and then overnight at room temperature. The
DCM solution was washed with brine (3 × 100 mL) and the organic
layer was dried over anhydrous MgSO4. After filtration of MgSO4, the
DCM solution was concentrated under the reduced pressure and the
product was isolated by crystallization from DCM/hexane/diethyl ether
mixture. The yield was 1.23 g (55%). The purity of the product was
2
European Polymer Journal 124 (2020) 109488
checked by a reversed-phase HPLC, showing a single peak with a re-
tention time of 4.0 min (UV detection at 230 nm). 1H NMR (300 MHz,
DMSO) δ ppm: 1.85 (s, 3H, eCH3), 3.05 (s, 1H, ^CH), 3.88 (d, 2H,
eCH2e), 5.37 and 5.68 (s, 1H, ]CH2), 8.37 (s, 1H, eNHe).
2-[1-Cyano-1-methyl-4-oxo-4-(2-thioxothiazolidin-3-yl)butylazo]-2-
methyl-5-oxo-5-(2-thioxothiazoli- din-3-yl)pentanenitrile (ACVA-(TT)2)
was prepared by the reaction of 4,4′-azobis(4-cyanovaleric acid)
(ACVA) with TT in tetrahydrofuran in the presence of N,N'-dicyclo-
hexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) as
described in [41].
Dithiobenzoic acid 1-cyano-1-methyl-4-oxo-4-(2-thioxothiazolidin-
3-yl)butyl ester (CTA-TT) was synthesized by the reaction of ACVA-
(TT)2 with bis(thiobenzoyl) disulfide in ethyl acetate at 353 K [41].
2.3. Synthesis of thermoresponsive diblock copolymers
The thermoresponsive diblock (A-B type) copolymers were synthe-
sized by RAFT polymerization technique in two synthetic steps [42].
First, the hydrophilic block A (PHPMA) was prepared by polymerizing
HPMA in the presence of CTA-TT and ACVA-TT. Then, block A was
subjected to a chain-extension polymerization with DEGMA in the
presence of AIBN to introduce the thermoresponsive block B. Three
different molar ratios of PHPMA to DEGMA were used to synthesize the
diblock copolymers with variable lengths of the thermo-responsive
blocks (PHPMA)53-b-(PDEGMA)26-78).
Example: A mixture of CTA-TT (22.1 mg, 58.2 μmol) and ACVA-TT
(14.1 mg, 29.1 μmol) was dissolved in DMSO (388 μL) and added to a
solution of HPMA (0.5 g, 3.49 mmol) in tert-butanol (3492 μL). The
reaction mixture was thoroughly bubbled with Ar and polymerized in
the sealed glass ampoules at 343 K for 16 h. The polymer was pre-
cipitated to a mixture of acetone and diethyl ether (3:1), re-dissolved in
methanol and purified by gel filtration using a SephadexTM LH-20 in
methanol. The methanolic solution was precipitated to diethyl ether
yielding 274.6 mg of the PHPMA hydrophilic polymer precursor
(PHPMA) as a pink powder.
Next, a mixture of PHPMA (20.0 mg, 2.61 μmol ~DTB groups) and
AIBN (0.09 mg, 0.52 μmol) was dissolved in 424 μL of dimethylaceta-
mide (DMAc), mixed with a solution of DEGMA (64.9 mg, 0.35 mmol)
in 424 μL of tert-butanol, thoroughly bubbled with Ar and polymerized
in the sealed glass ampoules at 343 K for 16 h. The diblock copolymer
was isolated by precipitation to diethyl ether yielding 65.0 mg of the
pale pink amorphous solid.
The diblock copolymer was dissolved in 650 μL DMAc, AIBN
(13.0 mg, 79.2 μmol) was added and the mixture was allowed to react
2 h at 353 K. After cooling the reaction mixture down to r.t., 1-ami-
nopropan-2-ol (6.5 mg, 86.5 μmol) was added and the mixture was
allowed to react for next 2 h. The resulting diblock copolymer was
precipitated to diethyl ether, re-dissolved in methanol and purified by
gel filtration using the Sephadex™ LH-20 in methanol. The methanolic
solution was precipitated to diethyl ether yielding 52.1 mg of the di-
block copolymer (PHPMA)53-b-(PDEGMA)78 as a white amorphous
solid. The SEC characteristics for the hydrophilic polymer precursor as
well as for the diblock copolymers are summarized in the Table 1.
2.4. Synthesis of thermoresponsive statistical copolymer
The termoresponsive statistical copolymer was prepared by copo-
lymerizing DEGMA with PGMA through the RAFT mechanism. A mix-
ture of DEGMA (0.5 g, 2.7 mmol), PGMA (36.4 mg, 0.3 mmol), CTA-
AIBN (2.0 mg, 8.9 μmol) and AIBN (0.3 mg, 1.8 μmol) was dissolved in
dioxan (738 μL), thoroughly bubbled with Ar and polymerized in the
sealed glass ampoules at 343 K for 4 h. The copolymer was precipitated
to diethyl ether, re-dissolved in methanol and purified by gel filtration
using a Sephadex™ LH-20 in methanol. The methanolic solution was
precipitated to diethyl ether yielding 240.8 mg of the statistical copo-
lymer as a pink waxy solid.
R. Konefał, et al.
Table 1
SEC, NMR and DLS characteristics for the hydrophilic polymer precursor (HP), thermoresponsive diblock copolymers (DB1 – DB3) and thermoresponsive statistical
copolymer (SP).
**
Sample (Co)polymer code DEGMA content (mol%)
PHPMA HP 0 7,660
P[(HPMA)53-b-(DEGMA)26] DB1 33
P[(HPMA)53-b-(DEGMA) 53] DB2 50
P[(HPMA)53-b-(DEGMA)78] DB3 60
P[(DEGMA)–co-(PGMA)] SP 93
* SEC.
** NMR.
*** DLS.
After that, the statistical copolymer was dissolved in 2.4 mL DMAc,
AIBN (21.2 mg, 0.13 mmol) was added and the mixture was allowed to
react 2 h at 353 K. The resulting product was precipitated to diethyl
ether, re-dissolved in methanol and purified by gel filtration using the
Sephadex™ LH-20 in methanol. The methanolic solution was pre-
cipitated to diethyl ether yielding 220.1 mg of the P[(DEGMA)–co-
(PGMA)] statistical copolymer as a white amorphous solid. The SEC
characteristics of the copolymer are stated in the Table 1.
2.5. UV–VIS spectrophotometry
The spectrophotometric analyses of the (co)polymers were carried
out in quartz glass cuvettes with an optical path length of 0.1 cm on a
UV–VIS spectrophotometer Specord Plus (Analytik Jena, Germany).
The molar content of dithiobenzoate (DTB) end group in the (co)
polymers were determined at 302 nm in methanol using the molar
absorption coefficient of 12,100 L/mol·cm. The molar content of the
terminal carbonylthiazolidine-2-thione reactive group in the (co)poly-
mers was determined at 305 nm in methanol using the molar absorption
coefficient of 10,300 L/mol·cm.
2.6. Size-exclusion chromatography (SEC)
The molecular weights and molecular weights distributions of the
(co)polymers were determined by SEC on a HPLC system (Shimadzu
VP, Japan), equipped with internal UV–VIS photodiode array detector,
and external differential refractive index and multiangle light scattering
detectors (Wyatt Technologies, USA). The TSK-Gel SuperAW3000 and
SuperAW4000 columns (6.0 × 150 mm, Tosoh Bioscience, Japan)
connected in series and 80% methanol/20% sodium acetate buffer
(0.3 M, pH 6.5) mixture as a mobile phase (flow rate 0.6 mL/min) was
used. A method based on the known total injected mass with an as-
sumption of 100% recovery was used for the estimation of the dn/dc
values needed for the calculation of the molecular weights from light
scattering data.
2.7. Dynamic light scattering (DLS)
The hydrodynamic diameter (DH) of the thermoresponsive copoly-
mers was measured by the DLS technique at a scattering angle θ = 173°
using a Nano-ZS instrument (Model ZEN3600, Malvern Instruments,
UK) equipped with a 632.8 nm laser. The temperature measurement
was performed to investigate the transition of the thermoresponsive
copolymer chains from random coils to polymer micelles in the tem-
perature interval 293–323 K (in 1 K increments) in PBS (1.0 mg/mL, pH
7.4) solutions. For the evaluation of the DLS data, the DTS(Nano)
program was used. The mean of at least three independent measure-
ments was calculated. The transition temperature (Ttr), characterizing
the polymer chain conformation changes, was evaluated from the
temperature dependence of the hydrodynamic diameter (DH) and was
determined from the intersection point of two lines formed by the linear
regression of a lower horizontal asymptote and a vertical section of the
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European Polymer Journal 124 (2020) 109488
***
*Mn [g/mol] *Mw/Mn DH310K [nm] ***
Ttr [K]
1.01 4.9 n.a.
11,600 1.20 5.6 312
17,100 1.20 29.5 308
21,500 1.30 33.8 304
37,320 1.09 1540.0 294
S-shaped curve (sigmoidal curve) fit.
2.8. NMR spectroscopy
Temperature dependences of 1H NMR spectra were acquired with
Bruker Avance III 600 spectrometer operating at 600.2 MHz. The width
of 90° pulse was 10 μs, relaxation delay 10 s, acquisition time 2.18 s, 16
scans. Each sample was kept for 15 min at the desired temperature
before measurement. The integrated intensities were determined with
spectrometer integration software with an accuracy of ± 1%. 2D 1H–1H
NOESY NMR spectra were recorded on the same spectrometer with
4098 Hz spectral window in f1 and f2 frequency axes, and mixing times
in the range 100–600 ms. A total of 16 scans was accumulated over
512 t1 (evolution time) increments with a relaxation delay of 10 s. The
temperature and time dependences of 1H spin-spin relaxation times T2
of HDO and selected proton groups the copolymer were measured using
the CPMG pulse sequence 90°x-(td-180°y-td)n-acquisition [43]. The re-
laxation delay between scans was 100 s, acquisition time 2.84 s with 2
scans. The relative error for T2 values did not exceed ± 5%. In all
measurements temperature was maintained constant within ± 0.2 K in
the range 290–330 K using a BVT 3000 temperature unit. Temperature
was calibrated using a standard 80% ethylene glycol (DMSO‑d6)
sample. All samples in D2O (Euriso-top, 99.9% of deuterium) solutions
(polymer concentrations c = 0.67 and 3 wt%) were filled into 5-mm
Norell NMR Tubes ST500-7 HT with ± 0.77 mm wall thickness and
178 mm length, which were degassed and sealed under nitrogen.
3. Results and discussion
3.1. Copolymer synthesis
All (co)polymers were generated using the controlled radical poly-
merization mechanism (RAFT technique) enabling the reproducible and
efficient preparation of highly defined materials with predetermined
molecular weights and high yield of terminal functional groups. The
hydrophilic polymer precursor (PHPMA) was synthesized by poly-
merizing HPMA in the presence of dithiobenzoate (DTB)-derived chain
transfer agent (CTA) yielding polymer with Mn of 7660 g/mol, narrow
molecular weights distribution (Mw/Mn = 1.01) and high functionality
of end groups (fDTB ≈ fTT ≈ 0.9). The hydrophilic polymer chain (block
A) was further extended with the thermoresponsive PDEGMA block
(block B). Three different ratios of DEGMA to PHPMA (44:1, 88:1 and
132:1) were used to synthesize the amphiphilic diblock copolymers
(PHPMA)-b-(PDEGMA) with the lengths of thermoresponsive block
ranging from approximately 4000–14,000 g/mol (see Table 1). The
gentle broadening in the molecular weight distributions of the diblock
copolymers can be ascribed to the presence of a low quantity of the
dead chains (not terminated with ~DTB groups) in the block A that
could not react further with DEGMA units through the RAFT me-
chanism. Both ~DTB and ~TT polymer end groups were blocked by a
homolytic reaction with a high molar excess of AIBN or by an amino-
lysis with a high molar excess of 1-aminopropan-2-ol, respectively, to
R. Konefał, et al.
Fig. 1. Scheme of synthesis of the hydrophilic polymer precursor (PHPMA) and the thermoresponsive diblock copolymers (PHPMA)-b-(DEGMA).
prevent their potential hydrolysis or other non-specific reactions during
next physico-chemical or biological experiments (for the reaction
scheme see Fig. 1).
The thermoresponsive statistical copolymer (P
[(DEGMA)–co–(PGMA)]) was prepared by copolymerizing DEGMA
with PGMA in the presence of dithiobenzoate (DTB)-derived chain
transfer agent (CTA) yielding polymer with Mn of 37,320 g/mol, narrow
molecular weights distribution (Mw/Mn = 1.09) and high functionality
of ~DTB end group (fDTB ≈ 1.0). The content of PGMA units in the
copolymer, determined using the 1H NMR, was 7.3 mol% (see Fig. S1 in
Supplementary material). The reactive PGMA co-monomer units were
incorporated to the copolymer due to the possibility of their future
modification with any azide group-containing compound through the
CuI-catalysed cycloaddition reaction (so called click chemistry). As in
the case of the diblock copolymers, terminal ~DTB group of the P
[(DEGMA)–co–(PGMA)] copolymer was removed by a homolytic re-
action with a high molar excess of the azoinitiator (for the reaction
scheme see Fig. 2).
3.2. Solution behavior of copolymers studied by DLS
The temperature-dependent changes in solution behavior of the
thermoresponsive diblock copolymers (DB1 – DB3) and thermo-
responsive statistical copolymer (SP) were studied by DLS in the solu-
tions mimicking physiological conditions. Specifically, we evaluated
how the thermoresponsive block (PDEGMA) length influences the
transition temperature (Ttr) of the diblock copolymers or how the
presence of the hydrophobic co-monomer (PGMA) influences the self-
assembly of the PDEGMA chains. We demonstrated that an increase in
molecular weight of the PDEGMA block (at a constant MW of the hy-
drophilic block) caused not only a decrease in transition temperatures
(Ttr) of the diblock copolymers but it also resulted in the formation of
larger-sized polymer coils (below Ttr) and to a certain extent in larger-
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European Polymer Journal 124 (2020) 109488
sized polymer micelles (above Ttr) (see Table 1 and Fig. 3). For ex-
ample, the DB1 copolymer with the MW ratio of the hydrophilic
(PHPMA) to the thermoresponsive (PDEGMA) blocks ~2/1 has 8 K
higher Ttr and forms 5 nm larger-sized micelles than the DB3 copolymer
having the MW of the hydrophobic block ~2 times higher than MW of
the hydrophilic block. It is a quite interesting finding showing that the
individual blocks of the diblock copolymers do not behave in the mi-
celles as independent structural units but they may influence each
other.
In the case of the statistical copolymer (SP), we observed that above
Ttr the copolymer chains assembled into hydrophobic globules with the
hydrodynamic size ~1500 nm. The reason for the formation of such
large-size and polydisperse objects (aggregates) instead of precisely
defined polymer micelles observed in the case of diblock copolymers is
absence of the hydrophilic blocks which condense and stabilize the
hydrophobic PDEGMA chains with a hydrophilic PHPMA. In an
agreement with the theory, statistical incorporation of the hydrophobic
PGMA units to the structure of the SP induced shift of Ttr to the lower
value (294 K) than it was measured for the unmodified PDEGMA
homopolymer (299 K) with the similar molecular weight.
3.3. Copolymers behavior on molecular level: 1H NMR spectra and fraction
p of proton groups (units) with significantly reduced mobility
Fig. 4 shows high-resolution 1H NMR spectra of a D2O solution
(c = 0.67 wt%) of the SP statistical copolymer measured under the
same instrumental conditions at three temperatures. The assignment of
resonances to various proton types is shown directly in the spectrum
measured at 290 K and chemical structure of statistical copolymer is
shown at the Figure. The broad signals “a” (δ ≈ 2 ppm) and “b”
(δ ≈ 1 ppm) are respectively related to methylene CH2 and methyl CH3
protons from the main chain of SP. DEGMA side chain C(O)OCH2 group
was assigned as “c” (δ ≈ 4.2 ppm) while peaks marked as “d, e, f”
R. Konefał, et al. European Polymer Journal 124 (2020) 109488

Fig. 2. Scheme of synthesis of the thermoresponsive P[(DEGMA)–co-(PGMA)] statistical copolymer.

Fig. 3. Temperature dependence of the hydrodynamic diameter (DH) of the thermoresponsive diblock copolymers (DB1 – DB3) (a) and statistical copolymer (SP) (b)
measured by DLS in PBS buffer (0.15 mM, pH 7.4) at a concentration c = 0.1 wt%.

(δ ≈ 3.9–3.6 ppm) correspond to OCH2 protons. Moreover strong signal
of OCH3 “g” is observed at δ = 3.45 ppm. Due to the hydrophobic
character and low content (7 mol%) signals related to PGMA monomer
repeating units are not visible in the spectrum (for chemical shifts see
spectrum in DMSO; Fig. S1 in Supplementary material). 1H NMR
spectra presented in Fig. 4 were measured at temperatures below the
LCST (290 K), in the middle of the transition (297 K) and above the
LCST (310 K) of SP. In this section we shall concentrate on changes in
integrated intensities of polymer signals. The most significant effect
observed in the spectra is a visible reduction in integral intensities and
disappearance of all signals of DEGMA units. This result is evidently
related to the fact that with increasing temperature, the mobility of the
part of polymer segments which form globular-like structures (me-
soglobules) decreases to such an extent that they escape detection in

5
R. Konefał, et al.
Fig. 4. 600.2 MHz 1H NMR spectra of SP statistical copolymer in D2O solution (c = 0.67 wt%) measured at 290, 297 and 310 K under the same instrumental
conditions.
high-resolution NMR spectra. It has been shown for other thermo-
responsive polymers studied by us and other authors previously that
proton linewidths of segments in globular structures are approx. 100
times larger and spin-spin relaxation times T2 are approx. 100 times
shorter in comparison with polymers segments retaining a high mobi-
lity [32,33]. This shows that mobility of polymer segments in globular
structures is affected in direction to solid-like systems. At the same time
linewidths and T2 values are for globules probably associated with
isotropic Brownian tumbling of globules as a whole as well as with
internal segmental mobility. Similar behavior as depicted in Fig. 4 was
previously observed also for PDEGMA homopolymer [44] and other
thermoresponsive polymer systems [13,28,32,33,38].
High-resolution 1H NMR spectra of the D2O solution (c = 0.67 wt%)
of the block copolymer DB2 recorded at three temperatures 295 K,
307 K and 320 K under the same instrumental conditions are presented
Fig. 5. 600.2 MHz 1H NMR spectra of DB2 block copolymer in D2O solution (c = 0.67 wt%) measured at 295, 307 and 320 K under the same instrumental conditions.
6
European Polymer Journal 124 (2020) 109488
in Fig. 5. Similarly as in Fig. 4 peaks assignments of the various proton
types are shown in the spectrum measured at 295 K. Signals of
PDEGMA units (a, b, c, d, e, f, g) are at the same positions as in the
spectra of statistical copolymer (SP cf. Fig. 4). Additional two peaks of
HPMA monomer repeating units side chain are detected: “k” (CH,
δ ≈ 3.9 ppm) and “j” (CH2, δ ≈ 3.3 ppm). Signals from remaining
HPMA proton groups (marked as a, b, l) are overlapped by DEGMA
backbone peaks. In the spectra recorded at 307 K and 320 K DEGMA
signals show similar effect as observed for SP, i.e., a decreasing integral
intensity of “a, b, d, e, f, g” signals and disapperance of “c” peak with
increasing temperature. Nevertheless, in comparison with SP copo-
lymer (cf. Fig. 4) this effect is much weaker and intensity changes de-
pend on distance of proton group from the main chain of the block;
increasing distance (i.e., increasing number of bonds from the back-
bone) - smaller change in the intensity: “c” fully disappears, “g” shows a
R. Konefał, et al. European Polymer Journal 124 (2020) 109488

certain broadening, which might be directly related to the hydro- we shall deal only with such signals which are not created by protons
phobicity of the respective proton groups [28,36]. On the other hand, from the both blocks (i.e., signals c, d + e + f, g, j, k). In comparison
intensity of peaks assigned to HPMA protons remains virtually un- with SP (Fig. 6a) a different temperature behavior of PDEGMA proton
changed. This effect in combination with DLS measurements imply groups is observed: only methylene protons “c” closest to block back-
nanoparticle formation at temperatures above the LCST. bone show a sharp transition with rather high pmax (≈0.80) value and
From temperature dependent integrated intensities of NMR signals LCST shifted to ≈305 K. Next OCH2 (d + e + f) side chain proton
it is possible to quantitatively characterize changes occurring during groups reach a smaller pmax (≈0.36) value at 310 K which with in-
the heating and cooling processes. For this purpose the values of the creasing temperature slightly drops. Moreover, methoxy end-group of
fraction p of proton groups of the given type with significantly reduced the side chain (g) exhibits a weak phase transition at higher tempera-
mobility were obtained using the relation [32,33,38,45,46]: ture (starting at 315 K) with pmax (≈0.20). On the other hand, inter-
esting behavior of the PHPMA block is observed: signals “j” and “k”.
I (T )
p=1− T0
The starting values of p-fraction of both PHPMA proton groups
I (T0) × T (1) (j ≈ 0.20 and k ≈ 0.30) decrease to reach minimum value at 306 K and
304 K, respectively, and then after a shallow minimum start to increase
where I(T) is the integrated intensity of given polymer signal in the
again at 315 K. The described behavior is in contrast to various ther-
spectrum at given absolute temperature T and I(T0) is the integrated
moresponsive homopolymers [PNIPAm, poly(N-iso-
intensity of this signal when no phase transition or other reason for the
propylmethacrylamide) (PNIPMAm), poly(N,N-diethylacrylamide),
reduced mobility of polymer segments occurs. For T0 we chose the
poly(vinyl methylether) (PVME), poly(N-vinyl caprolactam) (PVCL),
temperature where the integrated intensity of the given signal was the
poly(2-ethyl-2-oxazoline) (PEOx) [32,38,47], random or gradient co-
highest and therefore p(T0) = 0. Additionally, in denominator of the
polymers containing NIPAm or EOx [38,39] units as well to block co-
Eq. (1) we took into account the fact that the integrated intensities
polymers with PNIPAm or PEOx blocks [13,46] where temperature
should decrease with temperature as 1/T (Curie law holds also for
dependences of the p-fraction are virtually the same for all proton
nuclear magnetization). In Fig. 6a, temperature dependences of the p-
groups of the thermoresponsive component. In all these systems side
fraction of various proton types of SP in D2O solution (c = 0.67 wt%)
chains in thermoresponsive units are relatively short. On the other hand
are shown. For all proton groups (signal assignment in Fig. 4) p-fraction
a similar situation to described above by us for P[(HPMA)-b-(DEGMA)]
first slightly decreases and has a minimum at 293 K. At temperatures
diblock copolymers has been reported by Hiller et al. [48] who studied
above 293 K the values of the p-fraction are drastically increasing and
diblock copolymers composed of hydrophilic poly(2-methyl-2-oxazo-
phase transition occurs in accord with DLS results, (see Fig. 3b). Tem-
line) and hydrophobic poly(2-alkyl-2-oxazoline) where alkyl is pentyl,
perature dependences of the p-fraction determined from integrated in-
heptyl or nonyl, i.e. again systems where side chains are fairly long.
tensities of various DEGMA signals are for SP copolymer virtually the
Values of the p-fraction decreasing with temperature as observed for
same so confirming that p-fraction relates to PDEGMA units as a whole.
side chain proton groups of PHPMA units (signals j and k in Fig. 6b)
This means that all SP groups are similarly restricted in their mobility
resemble rather an UCST behavior like in κ-carrageenan [45] than
and the SP copolymer forms aggregates. Maximum values of the p-
normal temperature behavior of polymers in solution where mobility of
fraction (pmax ≈1) give quantitative information on the fraction of
polymer chains increases with temperature without any changes in p-
polymer chains which participate in the phase transition and have been
fraction values. Explanation of abovementioned effects is related to
achieved around 310 K. The LCST (defined as the temperature at pmax/
different changes in mobility of each proton groups. Mobility of
2) was for SP estimated as 298 K in agreement with DLS results when
PDEGMA block main chain decreasing with temperature caused by
we take into account the temperature in the middle of the transition
vanishing of hydrophilic interactions and their substitution by hydro-
interval (cf. Fig. 3b).
phobic interactions, affects also close methylene group (c). Side chain,
Temperature behavior of block copolymers was similarly char-
due to oxygen atoms, in some fraction (65% for d + e + f groups and
acterized by measuring the temperature dependences of their aqueous
80% for g methoxy group) remains hydrophilic. Temperature behavior
solutions. As an example temperature dependences of p-fraction of
of PHPMA block is connected with the PDEGMA block: mobility of
various proton types of DB3/D2O solution (c = 0.67 wt%) are pre-
PHPMA (j, k) is increasing to the phase transition temperature of
sented in Fig. 6b. Temperature dependences shown in the Fig. 6b are for
PDEGMA. In this temperature region copolymer chains are reorganizing
all measured signals (signal assignment in Fig. 5), but in the discussion

Fig. 6. Temperature dependences of the fraction p as determined for signals of various proton types in D2O solutions (c = 0.67 wt%) of SP (a) and DB3 (b) during
gradual heating.

7
R. Konefał, et al.
Fig. 7. Temperature dependences of the fraction p for the PDEGMA OCH2 protons (signal “c”) in D2O solutions (c = 0.67 wt%) of SP and DB1-DB3 copolymers (a)
and DB3 at two concentrations (c = 0.67 and 3 wt%) (b) during gradual heating.
and form nanoparticles. Similar behavior was observed also for other
two block copolymers (DB1 and DB2, cf. Fig. S2 in the Supplementary
material) and it is in good agreement with the DLS measurements (see
Fig. 3a).
For further considerations and comparisons as the most suitable
signal “c” of PDEGMA C(O)OCH2 protons was chosen. Temperature
dependences of the fraction p of D2O solutions (c = 0.67 wt%) of all
investigated polymers are shown in Fig. 7a. From Fig. 7a it follows that
in comparison with the SP (LCST = 298 K), in all block copolymers
values of the LCST are shifted to higher temperatures and depend on the
PDEGMA content in the copolymer with dependence: lower PDEGMA
content – higher LCST (DB1 = 310 K, DB2 = 308 K and DB3 = 305 K).
This result is in agreement with DLS results (Fig. 3) and with literature
[20,22,49,50]. Moreover, there is a visible change in the pmax values
which decrease with reduction of the PDEGMA content. These effects
are caused by changes in overall hydrophilicity of copolymer chains:
higher PHPMA content – higher hydrophilicity of the copolymer and
coil- to- globule transition becomes weaker due to smaller amount of
hydrophobic interactions above the LCST (shorter PDEGMA block)
[20]. Additionally, smaller pmax values which are directly connected to
mobility of the proton groups of the given type (here proton group “c”)
show that some percentage of them are still mobile (i.e. 40% for DB1),
but from DLS results (see Fig. 3a) it follows that nanoparticles are
formed. Therefore one can assume some hydration of polymer chains in
nanoparticles [51,52]. When nanoparticle is more hydrated, polymer
chains are more mobile and achieve smaller values of the pmax. Similar
behavior was observed for other PDEGMA proton groups and compar-
isons are shown in the Supplementary material, Figs. S3 and S4. From
studies based on cloud point or transmittance measurements it follows
that LCST of PDEGMA- based copolymers in aqueous solutions slightly
depends on polymer concentration [22,49]. For this reason we chose
DB3 copolymer as an example to study its solutions at two polymer
concentrations, c = 0.67 wt% and 3 wt%. Temperature dependences of
the p-fraction show similar temperature behavior for both polymer
concentrations for all copolymer proton types (see Fig. 6b and Fig. S5 in
the Supplementary material). Fig. 7b shows temperature dependences
of the fraction p in D2O solutions of the DB3 copolymer with polymer
concentrations c = 0.67 and 3 wt% recorded during gradual heating.
From Fig. 7b a slight shift of the LCST to smaller value (from 305 K to
304 K) with increasing polymer concentration is clearly visible. This
dependence is in agreement with abovementioned literature, as well as
with other thermoresponsive systems based on PNIPAM [12,13], PVCL
[47] or polyoxazolines [38]. Additionally, Fig. 7b shows that there is no
change in pmax values which were noticeably affected by copolymer
8
European Polymer Journal 124 (2020) 109488
composition.
Characterization of the reversibility of the phase transition after
heating process was obtained directly by similar measurements during
subsequent gradual cooling. During cooling process behavior of all
proton groups was similar to heating. Temperature dependences of the
fraction p of the PDEGMA OCH2 protons with significantly reduced
mobility (signal “c”) in D2O solutions (c = 0.67 wt%) of SP and DB3
during gradual heating and subsequent gradual cooling are shown in
Fig. 8a and b, respectively. In contrast to the cloud point measurements
presented in the literature [21,22,49], where hysteresis between
heating and cooling process was observed, in NMR measurements this
effect is not detected both in case of the SP and in diblock copolymer
solutions (for comparisons of the DB1, DB2 c = 0.67 wt% and DB3
c = 3 wt% see Figs. S6 and S7 in the Supplementary material).
Therefore the same way of the phase transition during heating and
cooling process indicates that on molecular level the phase transition of
all investigated samples is fully reversible without any hysteresis.
3.4. Behavior of water (HDO) and copolymer molecules as shown by spin-
spin relaxation times T2
Information on behavior of water and polymer-solvent interactions
(hydration) during phase-transition in aqueous solutions can be pro-
vided by NMR relaxation measurements of solvent, or in some cases
polymer molecules [53–55]. It was shown that especially spin-spin re-
laxation times T2 of water (HDO protons or D2O deuterons) are useful in
this respect [13,14,32,33,38]. Fig. 9a shows temperature dependence of
1
H spin-spin relaxation time T2 of HDO in D2O solution (c = 0.67 wt%)
of the SP. Measurements were done at temperatures based on the
temperature dependence of the p-fraction (see Fig. 6a). At all tem-
peratures there was a single line of HDO in 1H NMR spectrum and this
holds for all investigated samples. Low starting values of T2 (≈1 s) are
related to the relatively low mobility of water molecules and imply that
water molecules interact with polymer chains of the SP by hydrogen
bonding. Additionally, values of T2 slightly increase with temperature
which means that during the phase transition polymer - water hydrogen
bonding becomes weaker and HDO mobility somewhat increases. This
effect is also connected to formation of polymer aggregates when
polymer - polymer interactions outweigh: some water molecules are
slowly releasing and the other are still present in the globular structure.
This result is in contrast to results obtained for other thermoresponsive
polymer systems like PNIPAm, PNIPMAm or PVME where T2 values
were reduced at elevated temperatures [13,32,33]. In Fig. 9b time de-
pendence of 1H spin-spin relaxation time T2 of HDO in D2O solution
R. Konefał, et al.
Fig. 8. Temperature dependences of the fraction p of the PDEGMA OCH2 protons (signal “c”) with significantly reduced mobility in D2O solutions (c = 0.67 wt%) of
SP (a) and DB3 (b) during gradual heating and subsequent gradual cooling.
(c = 0.67 wt%) of the SP measured at 320 K (temperature above the
phase transition) is presented. A continuous increase of T2 values with
time is observed showing that portion of water molecules released from
aggregates increases with time a little. Moreover, no sedimentation of
SP aggregates was visually observed after the measurement. Almost the
same effect as shown in Fig. 9b was observed in PVCL [47] and PEO-b-
PNIPAM [13] aqueous solutions where releasing of originally bound
water with time starts directly without any induction period.
Fig. 10a shows temperature dependence of 1H spin-spin relaxation
time T2 of HDO in D2O solution (c = 0.67 wt%) of the DB3 block co-
polymer. Similarly to the SP sample measurements were done at tem-
peratures based on the temperature dependence of the p-fraction (see
Fig. 7a). In comparison with SP aqueous solution a different behavior is
observed. Firstly, much higher T2 value at temperature below the LCST
(T2 ≈ 8.5 s at 295 K) was measured which means that polymer - water
interactions are much weaker in comparison with SP solution. Sec-
ondly, T2 values significantly decrease with increasing temperature
which can be explained that some water molecules are hidden in na-
noparticle structures during their formation. Thirdly, at temperatures
above phase transition, the relaxation curves were bi-exponential and
two T2 components were necessary to fit well experimental relaxation
curves. This result shows the existence of two types of water at tem-
perature T = 330 K. First type is “free water” with longer relaxation
times (T2 ≈ 3 s) and second type is “bound” water, with T2 values
Fig. 9. Temperature dependence (a) and time dependence at 320 K (b) of 1H spin–spin relaxation times T2 of HDO in D2O solution (c = 0.67 wt%) of the SP.
9
European Polymer Journal 124 (2020) 109488
which are 3 orders of magnitude shorter (T2 ≈ 8 ms at 330 K), Similar
behavior was previously observed for PEOx homopolymers and PEOx-
based gradient or block copolymers [38,46] and in analogy with these
systems we assigned “free water” to HDO molecules in solution and
“bound water” to HDO molecules bound (confined) inside the nano-
particle. Intensities of the two T2 components show that the content of
the “bound” water is roughly 30%. The existence of two separate T2
components implies that exchange between “bound” and “free” water
molecules must be slow regarding T2 values. Fig. 10b shows time de-
pendences of two T2 components measured in the D2O solution of the
DB3 at 330 K. From Fig. 10b it follows that T2 values are virtually
constant for both types of water molecules showing that arrangement
with “free” and “bound” water is stable at least for 12 h. Comparable
behavior as shown in Fig. 10 was observed also with DB2 and DB1
copolymers as well as DB3 copolymer with concentration c = 3 wt%;
results are depicted in Figs. S8–S10 in the Supplementary material,
where Fig. S8 also illustrates marked differences in the fitting of ex-
perimental spin-spin relaxation curves at 295 K and 330 K.
In measurements of NMR relaxation times of polymer protons one
has to take into account the fact that in the LCST transition region and
above the transition these measurements provide information only on
the fraction of polymer protons (segments) (fraction 1 - p) which remain
mobile and therefore are directly detected in high-resolution NMR
spectra. Due to the fact that the p-fraction values of OCH3 group (signal
R. Konefał, et al.
Fig. 10. Temperature dependence (a) and time dependence at 330 K (b) of 1H spin-spin relaxation times T2 of HDO in D2O solution (c = 0.67 wt%) of the DB3.
Fig. 11. Temperature dependence (a) and time dependence at 330 K (b) of 1H spin-spin relaxation times T2 of OCH3 group “g” from PDEGMA block in D2O solution
(c = 0.67 wt%) of the DB3.
“g”) of the PDEGMA block are only slightly changing with temperature
(see Fig. S3b in the Supplementary material), 1H spin-spin relaxation
times T2 for this group were also measured. Obtained results for tem-
perature and time dependences for the DB3 block copolymer solution
(c = 0.67 wt%) are shown in Fig. 11. From Fig. 11a it follows that p-
fraction values for the OCH3 group “g” are directly connected with its
mobility. T2 are highest (and p-fraction is lowest) at the 300 K – tem-
perature where the phase transition starts. This behavior is directly
connected with the changes in polymer-water interactions during the
heating processes: at room temperature hydrogen bonds between water
and DEGMA units result in slightly reduced mobility of OCH3 group “g”,
next in pretransition region hydrogen bonding DEGMA-water becomes
weaker and mobility of the methoxy group increases and finally mo-
bility of the OCH3 group decreases in nanoparticle structure (but it
remains still relatively mobile that its detection in high-resolution in 1H
NMR spectra is possible). From 1H spin-spin relaxation time T2 time
dependence at 330 K (Fig. 11b), we see that T2 values remain virtually
unchanged for at least 12 h showing stability of this group in formed
nanoparticles. Similarly to water, analogous behavior as shown in
Fig. 11 was observed also with DB2 and DB1 copolymers (c = 0.67 wt
%) and DB3 copolymer with polymer concentration c = 3 wt% (see
Figs. S11–S13 in the Supplementary material).
By measuring 1H spin-spin relaxation time T2 of CH group (“k”)
10
European Polymer Journal 124 (2020) 109488
from the side chain of the PHPMA block it is possible to follow changes
in mobility of hydrophilic part of the copolymer during the phase
transition. Temperature and time dependence of 1H spin-spin relaxation
time T2 of the “k” CH group in D2O solution (c = 0.67 wt%) of the DB3
block copolymer are shown in Fig. 12. Similarly to the PDEGMA OCH3
“g” group, p-fraction values of the PHPMA CH “k” group (Fig. 6b) are
also directly connected with its T2 values (mobility). Fig. 12a shows
significantly increasing T2 values with increasing temperature. They are
caused by temperature as well as by changes in interactions in solution,
PDEGMA block exhibits the phase transition and gives more space and
less spherical restrictions to the PHPMA block. On the other hand,
during first 6 h above the phase transition T2 values of the “k” CH group
decrease and imply some changes in the behavior of the PHPMA block,
indicating probable changes in interactions in nanoparticles. Similar
results as shown in Fig. 12 were obtained also with DB2 and DB1 co-
polymers and DB3 copolymer with concentration c = 3 wt% (cf. Figs.
S14–S16 in the Supplementary material).
3.5. Conformational changes of block copolymer: 2D 1H–1H NOESY NMR
spectra
To understand the changes occurring during the phase separation
and obtain information on spatial proximity between proton groups of
R. Konefał, et al. European Polymer Journal 124 (2020) 109488

Fig. 12. Temperature dependence (a) and time dependence at 330 K (b) of 1H spin-spin relaxation time T2 of the PHPMA group “k” in D2O solution (c = 0.67 wt%) of
the DB3.

Fig. 13. 2D NOESY spectrum of DB3 block copolymer in D2O solution (c = 3 wt%) measured at 295 K with mixing time 600 ms. On the right up there is expanded
part of the spectrum close to PHPMA “k” group cross-peaks, on the right down there is 1D slice spectrum extracted from the PHPMA “k” signal of the NOESY
spectrum.

PDEGMA and PHPMA units, 2D nuclear Overhauser effect spectroscopy
(NOESY) was employed. From NOESY NMR it is possible to obtain in-
formation on the spatial interactions of different nuclear spins in dis-
tances to maximum 0.5 nm [56–58]. Experimental parameters (espe-
cially the mixing time) used in the NOESY NMR measurements were
carefully chosen based on our previous studies of other thermo-
responsive polymer systems [13,35,38] as well as studies of other au-
thors [56,58]. DB3 (c = 3 wt%) D2O solution was chosen for that
purpose and 2D 1H–1H NOESY NMR spectra were measured at four
temperatures: at 295 K (starting temperature, below the transition),
300 K (temperature directly below the transition), 304 K (in the middle
of the transition) and 315 K (above the transition). We assumed to use
the fact that even at 315 K the value of the p-fraction of the PDEGMA
OCH3 group “g” is low (pmax = 0.09, cf. Fig. 6b) and therefore a major
part of these side chain end-groups is directly detected in high-resolu-
tion NMR spectra at this temperature. In NOESY spectrum measured at
295 K (Fig. 13) we detected not only cross-peaks between various
proton groups within PDEGMA or PHPMA units, but also weaker cross-
peaks between side chain CH (“k”) protons of PHPMA units (signal at
3.93 ppm) and PDEGMA side chain protons (Fig. 13 on the right down).
The presence of all these cross-peaks implies that distances between
respective protons are smaller than 0.5 nm. PDEGMA and PHPMA units
which are in close proximity can be both from the same chain of the
copolymer, assuming a random-coil conformation of copolymer chains
at room temperature, and from different copolymer chains. 2D NOESY
spectra with 1D slices for other temperatures are shown in the
Supplementary material (Figs. S17–S19). Fig. 14 then shows tempera-
ture dependences of the integrated intensities of signals in 1D slices,
extracted from the signal of CH protons (“k”) of PHPMA units of the 2D
NOESY spectra measured with the mixing time 600 ms for D2O solution
of the DB3 diblock copolymer. Dependences of the respective in-
tegrated intensities in 1D slices extracted from NOESY spectra mea-
sured with mixing time 100 ms were similar, only the signal/noise ratio
in these slices was somewhat lower. This is consistent with build-up
curves (measured in 259 K) which show that in the range 100–600 ms
intensities of the crosspeaks increased with the mixing time as illu-
strated in Fig. S20 in the Supplementary material. Due to the fact that
distance between CH (“k”) and CH2 (“j”) PHPMA protons cannot
change, cross-peak between these groups was used as internal standard
with integral intensity 1. From the Fig. 14 it follows that unexpectedly

11
R. Konefał, et al.
integral intensity of cross-peaks between CH (“k”) protons of PHPMA
and PDEGMA side chain protons (signals “d + e + f” as well as “g”)
increases with temperature. This result excludes typical core- to- shell
formation of nanoparticles. Moreover, these findings, together with
values of the p-fraction and T2 values mentioned above, suggest that
above the phase transition temperature block copolymer chains create
multi-chain aggregates with partially hydrated (PHPMA, PDEGMA side
chains) and dehydrated (PDEGMA main chain) volumes (parts) in their
nanostructures. Similar result was obtained also from the 2D NOESY
spectra measured with the mixing time 100 ms.
4. Conclusions
In the present study we report on temperature behavior of ther-
moresponsive PDEGMA-based statistical and diblock copolymers in
D2O solutions as investigated by 1H NMR methods which were com-
pared with DLS measurements. Measurements of 1H NMR spectra, 1H
spin-spin relaxation time T2 and 2D 1H–1H NOESY spectra were used for
the characterization of structural changes on molecular level and be-
havior of water and copolymer molecules during the temperature-in-
duced phase transition. In comparison with other thermoresponsive
polymers (PNIPAm, PNIPMAm, PVME, PVCL, PEOx) some important
differences were revealed for PDEGMA copolymers.
P[(DEGMA)–co-(PGMA)] statistical copolymer (SP) and three
(PHPMA)-b-(PDEGMA) (DB1-3) block copolymers (with constant
length of PHPMA block: 53 monomer units and different lengths of
PDEGMA blocks: 26; 53 and 78 monomer units) were studied. In the
transition region, we observed at similar temperatures an increase in
the hydrodynamic sizes of the respective nanoparticles, as determined
by lower diffusion of the particles in DLS experiments and fraction p of
proton groups (units in the case of the SP copolymer) with significantly
reduced mobility in NMR experiments. Temperature dependences of
the p-fraction show that for D2O solution of the SP the phase transition
is relatively sharp with a slight difference between backbone and side
chain. In contrast to the SP, the phase separation of DB in D2O solutions
occurs in PDEGMA block backbone and in its nearest methylene group.
Values of the p-fraction of PDEGMA side chain proton groups decrease
with increasing distance from the backbone. Moreover, transition
temperatures substantially depend on polymer composition in accord
with DLS results and slightly on the concentration of the solution. The
temperature dependences of the p-fraction measured during gradual
cooling after preceding gradual heating show that for all samples the
phase transition is fully reversible on the molecular level.
From measurements of 1H spin-spin relaxation times T2 of HDO, a
12
European Polymer Journal 124 (2020) 109488
Fig. 14. Chemical structure and inter-
molecular correlations in DB3 block copo-
lymer (left). Temperature dependences of
integrated intensities of various signals in
1D slices extracted from the signal of CH
protons (“k”) of PHPMA units (at 3.93 ppm)
of the NOESY NMR spectra of D2O solution
(c = 3 wt%) of the DB3 copolymer (right).
significantly different behavior of the water molecules in SP and DB
solutions was observed. While in case of SP solution the single T2 values
increase with temperature, in case of DB solutions two types of water
molecules –“free” and “bound” - with long and very short T2 values,
respectively, were detected at temperatures above the transition. The T2
values of the two types of water remain stable for at least 12 h at 330 K
and no release of the “bound” water was observed during this time. The
existence of two T2 components indicates that the exchange between
“bound” and “free” water must be slow relative to T2 values. In case of
the DB copolymers, T2 values were also measured for polymer side
chain groups of both PDEGMA and PHPMA blocks. In case of PDEGMA,
OCH3 group was chosen due to the small values of the p-fraction. Its T2
values decrease with temperature, indicating that mobility of this group
is reduced in the form of nanoparticles. For PHPMA side chain CH
group, a different temperature effect is observed: T2 values increase
with temperature, which is normal behavior of hydrophilic polymers in
water, but at temperature above the phase transition they decrease with
time because mobility of the hydrophilic block is reduced due to
polymer-polymer interactions in nanoparticles.
2D 1H–1H NOESY NMR spectra show that in case of DB3 block
copolymer in D2O solution (c = 3 wt%) some HPMA and DEGMA units
are in close contact already at room temperature. As temperature in-
creases in some NOESY cross-peaks between the PHPMA and PDEGMA
side chains their integral intensity increases. Based on these facts, we
assume that polymer chains are closer to each other in nanoparticles
and therefore core-to-shell morphology is improbable in these systems.
It is expected that the results reported herein will assist in designing of
“smart” thermoresponsive polymer drug delivery systems.
CRediT authorship contribution statement
Rafał Konefał: Conceptualization, Formal analysis, Investigation,
Writing - original draft. Jiří Spěváček: Writing - review & editing,
Supervision, Funding acquisition. Gabriela Mužíková: Investigation.
Richard Laga: Formal analysis, Investigation, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
R. Konefał, et al.
Acknowledgement
Support by the Czech Science Foundation (Project 15-13853S) and
by the Ministry of Education, Youth and Sports of the Czech Republic
(National Sustainability Program II – project BIOCEV-FAR LQ1604 and
INTER-EXCELLENCE/INTER-ACTION program – project LTAUSA18173
is gratefully acknowledged.
Data availability statement
The raw/processed data required to reproduce these findings cannot
be shared at this time as the data also form part of an ongoing study.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.eurpolymj.2020.109488.
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