Thermoresponsive behavior of poly(DEGMA)-based copolymers. NMR and dynamic light scattering study of aqueous solutions

European Polymer Journal 124 (2020) 109488

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Thermoresponsive behavior of poly(DEGMA)-based copolymers. NMR and dynamic light scattering study of aqueous solutions ⁎

Rafał Konefał , Jiří Spěváček

T

⁎,1

, 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: Thermoresponsive polymer Poly(2-(2-methoxyethoxy)ethyl methacrylate) (PDEGMA) PHPMA Aqueous solution NMR Spin-spin relaxation times T2 NOESY Dynamic light scattering

We studied temperature behavior of diblock copolymers of poly[2-(2-methoxyethoxy)ethyl methacrylate] (PDEGMA) with poly(N-(2-hydroxypropyl)methacrylamide and a statistical copolymer PDEGMA-co-N-propargylmethacrylamide in aqueous solutions. NMR spectra and dynamic light scattering showed that the copolymers exhibit a reversible phase transition which depends on the composition of the block copolymer and slightly on polymer concentration. In contrast to statistical copolymer, different behavior of proton groups in polymer backbone (and in the nearest side chain OCH2 group) on the one hand and in other side chain groups of the PDEGMA on the other hand was observed for diblock copolymers. Additionally, two types of water molecules were detected in solutions of the diblock copolymers above the phase transition –“free” with long and “bound” 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 copolymers in solution on the molecular level and provides important information for the design of “smart” thermoresponsive polymer-based drug delivery systems.

1. Introduction 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, thermoresponsive 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 delivery purposes, since the polymers having LCST values in this temperature 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 physiological 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-

isopropylacrylamide) (PNIPAm) with LCST around 305 K [12–15]. 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 ethylene glycol units, and poly(2-[2-(2- methoxyethoxy)ethoxy] ethyl methacrylate) (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 copolymerizing OEGMA with various hydrophobic comonomers. Moreover, 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 applications. In aqueous media, block copolymers where one component is hydrophilic and the other thermoresponsive can form various self-

⁎

Corresponding authors. E-mail addresses: [email protected] (R. Konefał), [email protected], [email protected] (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 Published by Elsevier Ltd.

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checked by a reversed-phase HPLC, showing a single peak with a retention 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]-2methyl-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'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) as described in [41]. Dithiobenzoic acid 1-cyano-1-methyl-4-oxo-4-(2-thioxothiazolidin3-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].

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 excellent 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, infrared (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 techniques were successfully applied to study various single- and multicomponent 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 (applied 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 thermoresponsive 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 copolymers are suitable for application in biomedicine and/or drug and gene delivery systems.

2.3. Synthesis of thermoresponsive diblock copolymers The thermoresponsive diblock (A-B type) copolymers were synthesized 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 precipitated 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 dimethylacetamide (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-aminopropan-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 diblock 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. 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 (CTAAIBN); N,N'-dicyclohexylcarbodiimide (DCC); 4-dimethylaminopyridine (DMAP); N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC); magnesium sulfate; methacrylic acid; methacryloyl chloride; 2-(2-methoxyethoxy)ethyl methacrylate (DEGMA); propargylamine; sodium carbonate and thiazolidine-2-thione (TT) were purchased from Sigma-Aldrich, Czech Republic. All solvents used in this work were of high-purity grade with extremely low water levels purchased 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 dichloromethane in the presence of sodium carbonate according to procedure 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 hydrochloride (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

2.4. Synthesis of thermoresponsive statistical copolymer The termoresponsive statistical copolymer was prepared by copolymerizing DEGMA with PGMA through the RAFT mechanism. A mixture of DEGMA (0.5 g, 2.7 mmol), PGMA (36.4 mg, 0.3 mmol), CTAAIBN (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 copolymer as a pink waxy solid. 2

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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

**

*Mn [g/mol]

*Mw/Mn

***

PHPMA P[(HPMA)53-b-(DEGMA)26] P[(HPMA)53-b-(DEGMA) 53] P[(HPMA)53-b-(DEGMA)78] P[(DEGMA)–co-(PGMA)]

HP DB1 DB2 DB3 SP

0 33 50 60 93

7,660 11,600 17,100 21,500 37,320

1.01 1.20 1.20 1.30 1.09

4.9 5.6 29.5 33.8 1540.0

DEGMA content (mol%)

DH310K [nm]

***

Ttr [K]

n.a. 312 308 304 294

* SEC. ** NMR. *** DLS.

S-shaped curve (sigmoidal curve) fit.

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 precipitated 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.

2.8. 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 relaxation 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.

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)polymers 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 assumption 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.

3. Results and discussion 3.1. Copolymer synthesis All (co)polymers were generated using the controlled radical polymerization 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 polymerizing 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 mechanism. Both ~DTB and ~TT polymer end groups were blocked by a homolytic reaction with a high molar excess of AIBN or by an aminolysis with a high molar excess of 1-aminopropan-2-ol, respectively, to

2.7. Dynamic light scattering (DLS) The hydrodynamic diameter (DH) of the thermoresponsive copolymers 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 temperature 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 measurements 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 3

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Fig. 1. Scheme of synthesis of the hydrophilic polymer precursor (PHPMA) and the thermoresponsive diblock copolymers (PHPMA)-b-(DEGMA).

sized polymer micelles (above Ttr) (see Table 1 and Fig. 3). For example, 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 micelles 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.

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 reaction with a high molar excess of the azoinitiator (for the reaction scheme see Fig. 2). 3.2. Solution behavior of copolymers studied by DLS

3.3. Copolymers behavior on molecular level: 1H NMR spectra and fraction p of proton groups (units) with significantly reduced mobility

The temperature-dependent changes in solution behavior of the thermoresponsive diblock copolymers (DB1 – DB3) and thermoresponsive statistical copolymer (SP) were studied by DLS in the solutions 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 selfassembly of the PDEGMA chains. We demonstrated that an increase in molecular weight of the PDEGMA block (at a constant MW of the hydrophilic 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-

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” 4

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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 (mesoglobules) decreases to such an extent that they escape detection in 5

European Polymer Journal 124 (2020) 109488

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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.

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 copolymer (cf. Fig. 4) this effect is much weaker and intensity changes depend on distance of proton group from the main chain of the block; increasing distance (i.e., increasing number of bonds from the backbone) - smaller change in the intensity: “c” fully disappears, “g” shows a

high-resolution NMR spectra. It has been shown for other thermoresponsive 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 mobility [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

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we shall deal only with such signals which are not created by protons from the both blocks (i.e., signals c, d + e + f, g, j, k). In comparison with SP (Fig. 6a) a different temperature behavior of PDEGMA proton groups is observed: only methylene protons “c” closest to block backbone show a sharp transition with rather high pmax (≈0.80) value and LCST shifted to ≈305 K. Next OCH2 (d + e + f) side chain proton groups reach a smaller pmax (≈0.36) value at 310 K which with increasing temperature slightly drops. Moreover, methoxy end-group of the side chain (g) exhibits a weak phase transition at higher temperature (starting at 315 K) with pmax (≈0.20). On the other hand, interesting behavior of the PHPMA block is observed: signals “j” and “k”. The starting values of p-fraction of both PHPMA proton groups (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 again at 315 K. The described behavior is in contrast to various thermoresponsive homopolymers [PNIPAm, poly(N-isopropylmethacrylamide) (PNIPMAm), poly(N,N-diethylacrylamide), poly(vinyl methylether) (PVME), poly(N-vinyl caprolactam) (PVCL), poly(2-ethyl-2-oxazoline) (PEOx) [32,38,47], random or gradient copolymers containing NIPAm or EOx [38,39] units as well to block copolymers with PNIPAm or PEOx blocks [13,46] where temperature dependences of the p-fraction are virtually the same for all proton groups of the thermoresponsive component. In all these systems side chains in thermoresponsive units are relatively short. On the other hand a similar situation to described above by us for P[(HPMA)-b-(DEGMA)] diblock copolymers has been reported by Hiller et al. [48] who studied diblock copolymers composed of hydrophilic poly(2-methyl-2-oxazoline) and hydrophobic poly(2-alkyl-2-oxazoline) where alkyl is pentyl, heptyl or nonyl, i.e. again systems where side chains are fairly long. Values of the p-fraction decreasing with temperature as observed for side chain proton groups of PHPMA units (signals j and k in Fig. 6b) resemble rather an UCST behavior like in κ-carrageenan [45] than normal temperature behavior of polymers in solution where mobility of polymer chains increases with temperature without any changes in pfraction values. Explanation of abovementioned effects is related to different changes in mobility of each proton groups. Mobility of PDEGMA block main chain decreasing with temperature caused by vanishing of hydrophilic interactions and their substitution by hydrophobic interactions, affects also close methylene group (c). Side chain, due to oxygen atoms, in some fraction (65% for d + e + f groups and 80% for g methoxy group) remains hydrophilic. Temperature behavior of PHPMA block is connected with the PDEGMA block: mobility of PHPMA (j, k) is increasing to the phase transition temperature of PDEGMA. In this temperature region copolymer chains are reorganizing

certain broadening, which might be directly related to the hydrophobicity of the respective proton groups [28,36]. On the other hand, intensity of peaks assigned to HPMA protons remains virtually unchanged. This effect in combination with DLS measurements imply nanoparticle formation at temperatures above the LCST. From temperature dependent integrated intensities of NMR signals it is possible to quantitatively characterize changes occurring during the heating and cooling processes. For this purpose the values of the fraction p of proton groups of the given type with significantly reduced mobility were obtained using the relation [32,33,38,45,46]:

p=1−

I (T ) I (T0) ×

T0 T

(1)

where I(T) is the integrated intensity of given polymer signal in the spectrum at given absolute temperature T and I(T0) is the integrated intensity of this signal when no phase transition or other reason for the reduced mobility of polymer segments occurs. For T0 we chose the temperature where the integrated intensity of the given signal was the highest and therefore p(T0) = 0. Additionally, in denominator of the Eq. (1) we took into account the fact that the integrated intensities should decrease with temperature as 1/T (Curie law holds also for nuclear magnetization). In Fig. 6a, temperature dependences of the pfraction of various proton types of SP in D2O solution (c = 0.67 wt%) are shown. For all proton groups (signal assignment in Fig. 4) p-fraction first slightly decreases and has a minimum at 293 K. At temperatures above 293 K the values of the p-fraction are drastically increasing and phase transition occurs in accord with DLS results, (see Fig. 3b). Temperature dependences of the p-fraction determined from integrated intensities of various DEGMA signals are for SP copolymer virtually the same so confirming that p-fraction relates to PDEGMA units as a whole. This means that all SP groups are similarly restricted in their mobility and the SP copolymer forms aggregates. Maximum values of the pfraction (pmax ≈1) give quantitative information on the fraction of polymer chains which participate in the phase transition and have been achieved around 310 K. The LCST (defined as the temperature at pmax/ 2) was for SP estimated as 298 K in agreement with DLS results when we take into account the temperature in the middle of the transition interval (cf. Fig. 3b). Temperature behavior of block copolymers was similarly characterized by measuring the temperature dependences of their aqueous solutions. As an example temperature dependences of p-fraction of various proton types of DB3/D2O solution (c = 0.67 wt%) are presented in Fig. 6b. Temperature dependences shown in the Fig. 6b are for 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

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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.

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.

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 comparisons 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

3.4. Behavior of water (HDO) and copolymer molecules as shown by spinspin relaxation times T2 Information on behavior of water and polymer-solvent interactions (hydration) during phase-transition in aqueous solutions can be provided by NMR relaxation measurements of solvent, or in some cases polymer molecules [53–55]. It was shown that especially spin-spin relaxation 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 temperatures 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 dependence of 1H spin-spin relaxation time T2 of HDO in D2O solution 8

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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.

which are 3 orders of magnitude shorter (T2 ≈ 8 ms at 330 K), Similar behavior was previously observed for PEOx homopolymers and PEOxbased 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 nanoparticle. 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 dependences 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 experimental 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

(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-bPNIPAM [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 copolymer. Similarly to the SP sample measurements were done at temperatures 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. Secondly, T2 values significantly decrease with increasing temperature which can be explained that some water molecules are hidden in nanoparticle 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 temperature 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

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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 temperature and time dependences for the DB3 block copolymer solution (c = 0.67 wt%) are shown in Fig. 11. From Fig. 11a it follows that pfraction values for the OCH3 group “g” are directly connected with its mobility. T2 are highest (and p-fraction is lowest) at the 300 K – temperature 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 mobility 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”)

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 copolymers 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 10

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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.

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 temperature 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 integrated intensities in 1D slices extracted from NOESY spectra measured 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 illustrated 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

PDEGMA and PHPMA units, 2D nuclear Overhauser effect spectroscopy (NOESY) was employed. From NOESY NMR it is possible to obtain information on the spatial interactions of different nuclear spins in distances to maximum 0.5 nm [56–58]. Experimental parameters (especially the mixing time) used in the NOESY NMR measurements were carefully chosen based on our previous studies of other thermoresponsive polymer systems [13,35,38] as well as studies of other authors [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-resolution 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 crosspeaks 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). 11

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Fig. 14. Chemical structure and intermolecular correlations in DB3 block copolymer (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 increases 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.

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 thermoresponsive PDEGMA-based statistical and diblock copolymers in D2O solutions as investigated by 1H NMR methods which were compared 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 behavior of water and copolymer molecules during the temperature-induced 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

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 influence the work reported in this paper.

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Acknowledgement [21]

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.

[22]

[23]

Data availability statement

[24]

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.

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Appendix A. Supplementary material [26]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2020.109488.

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