Alternative to Poly(2-Isopropyl-2-Oxazoline) with a Reduced Ability to Crystallize and Physiological LCST

International Journal of Molecular Sciences

Communication Alternative to Poly(2-isopropyl-2-oxazoline) with a Reduced Ability to Crystallize and Physiological LCST

Wojciech Wałach 1 , Agnieszka Klama-Baryła 2, Anna Sitkowska 2, Agnieszka Kowalczuk 1 and Natalia Oleszko-Torbus 1,*

1 Centre of Polymer and Carbon Materials, Polish Academy of Sciences, 34 M. Curie-Skłodowskiej St., 41-819 Zabrze, Poland; [email protected] (W.W.); [email protected] (A.K.) 2 Dr. Stanislaw Sakiel Center for Burn Treatment, 2 Jana Pawla II St., 41-100 Siemianowice Slaskie, Poland; [email protected] (A.K.-B.); [email protected] (A.S.) * Correspondence: [email protected]; Tel.: +48-32-271-60-77 (ext. 248)

Abstract: In this work, we sought to examine whether the presence of alkyl substituents randomly distributed within the main chain of a 2-isopropyl-2-oxazoline-based copolymer will decrease its ability to crystallize when compared to its homopolymer. At the same time, we aimed to ensure an appropriate hydrophilic/lipophilic balance in the copolymer and maintain the phase transition in the vicinity of the human body temperature. For this reason, copolymers of 2-ethyl-4-methyl- 2-oxazoline and 2-isopropyl-2-oxazoline were synthesized. The thermoresponsive behavior of the copolymers in water, the influence of salt on the cloud point, the presence of hysteresis of the phase transition and the crystallization ability in a water solution under long-term heating conditions were studied by turbidimetry. The ability of the copolymers to crystallize in the solid state, and their thermal properties, were analyzed by differential scanning calorimetry and X-ray diffractometry. A cytotoxicity assay was used to estimate the viability of human fibroblasts in the presence of the obtained polymers. The results allowed us to demonstrate a nontoxic alternative to poly(2-isopropyl- Citation: Wałach, W.; Klama-Baryła, 2-oxazoline) (PiPrOx) with a physiological phase transition temperature (LCST) and a greatly reduced A.; Sitkowska, A.; Kowalczuk, A.; tendency to crystallize. The synthesis of 2-oxazoline polymers with such well-defined properties is Oleszko-Torbus, N. Alternative to important for future biomedical applications. Poly(2-isopropyl-2-oxazoline) with a Reduced Ability to Crystallize and Keywords: poly(2-isopropyl-2-oxazoline); crystallization; thermal properties; LCST Physiological LCST. Int. J. Mol. Sci. 2021, 22, 2221. https://doi.org/ 10.3390/ijms22042221 1. Introduction Received: 29 January 2021 Accepted: 20 February 2021 Stimuli-responsive polymers showing a reversible response to external stimuli have Published: 23 February 2021 attracted much attention as “smart” and advanced materials in the last few decades [1]. In particular, thermoresponsive polymers exhibiting a phase transition around body tem- Publisher’s Note: MDPI stays neutral perature are of special interest due to their potential for biomedical applications. Among with regard to jurisdictional claims in thermoresponsive polymers with a peptidomimetic structure, exhibiting a lower criti- published maps and institutional affil- cal solution temperature (LCST) at the physiological level, poly(N-isopropylacrylamide) iations. (PNIPAM) and poly(2-isopropyl-2-oxazoline) (PiPrOx) are widely known. PNIPAM is considered the “gold standard” of LCST polymers for biomedical applications [2]. The interesting property of this polymer is the low sensitivity of its LCST to environmental conditions (variations in pH, concentration or the chemical environment affect the LCST of

Copyright: © 2021 by the authors. PNIPAM by only a few degrees [3,4]). When heated, the PNIPAM aqueous solution exhibits Licensee MDPI, Basel, Switzerland. a sharp phase transition; however, an undesired broad hysteresis can be observed during This article is an open access article cooling, which is involved with the irreversible coil-to-globule transition [5]. PiPrOx is distributed under the terms and the structural isomer of PNIPAM, and it exhibits no hysteresis of the phase transition [6,7]. conditions of the Creative Commons PiPrOx is a member of a large group of polymers known as poly(2-oxazoline)s that are Attribution (CC BY) license (https:// obtained via cationic ring opening polymerization (CROP) of ﬁve-membered cyclic imino creativecommons.org/licenses/by/ ethers containing a double bond at position 2 (Figure1)[8]. 4.0/).

Int. J. Mol. Sci. 2021, 22, 2221. https://doi.org/10.3390/ijms22042221 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2221 2 of 11 Int. J. Mol. Sci. 2021, 22, 2221 2 of 11

R N 2 R2 R3 3 4 CROP N CH CH R1 2 1 5 C O O R 3 R1 FigureFigure 1.1. TheThe 2-Oxazoline2-Oxazoline polymerizationpolymerization scheme.scheme.

TheThe naturenature ofof substituentssubstituents RR11,R, R22 and R 3 determinesdetermines the the poly(2 poly(2-oxazoline)-oxazoline) properties. PiPrOxPiPrOx withwith thethe isopropylisopropyl substituentsubstituent RR11 isis knownknown toto bebe aa thermoresponsivethermoresponsive polymerpolymer [[9]9] exhibitingexhibiting nontoxicity nontoxicity toward toward a variety a variety of cell of linescell lines [10–13 [10] and–13] thus and is thus discussed is discussed as an alterna- as an tivealternative to PNIPAM. to PNIPAM. The dependence The dependence of the phase of the transition phase temperaturetransition temperature on the molar on mass the ormolar the presencemass or ofthe a surfactantpresence of was a s foundurfactant to be was more found pronounced to be more for PiPrOx pronounced than for for PNIPAM PiPrOx [7 than,14]. Anotherfor PNIPAM unique [7,14] feature. Another of PiPrOx unique is its feature tendency of to PiPrOx crystallize is its upon tendency the prolonged to crystallize incubation upon atthe an prolonged elevated temperatureincubation at and an elevated the formation temperature of hierarchically and the formation structured of objects hierarchically [15–17]. However,structured this objects property [15– seems17]. However, detrimental this for property certain, mainlyseems biomedical,detrimental applications. for certain, mainly Hence, thebiomedical, goal of the applications. present study Hence, was to the obtain goal anof the alternative present tostudy PiPrOx was with to obtain a reduced an alternative ability to crystallize.to PiPrOx with It was a reduced important ability to carry to crystallize. out the modification It was important so that theto carry obtained out polymerthe modifica- was nontoxiction so that and the exhibited obtained a LCST polymer close was tothe nontoxic human and body exhibited temperature. a LCST close to the human bodyThe temperature. chemical structure of PiPrOx (with the isopropyl substituent R1 attached to the planar amide groups) favors easy packing and, as a consequence, ordering of the polymer The chemical structure of PiPrOx (with the isopropyl substituent R1 attached to the chains.planar amide Crystallization groups) favors of homo- easy and packing copolymers and, as of a 2-isopropyl-2-oxazolineconsequence, ordering of (iPrOx) the polymer in the solid-statechains. Crystallization [18], in aqueous of homo solutions- and copolymers [15,17,19–26 of] and 2-isopropyl in organic-2- [oxazoline27] and water/organic (iPrOx) in the mixturessolid-state [16 [18]] has, in been aqueous described solutions in great [15,17,19 detail.–26] Efforts and in were organic made [27] to and reduce water/organic the ability of PiPrOx to crystallize. For that purpose, the introduction of additional 2-oxazoline mixtures [16] has been described in great detail. Efforts were made to reduce the ability of comonomers with R substituents other than isopropyl was carried out to disrupt the PiPrOx to crystallize.1 For that purpose, the introduction of additional 2-oxazoline comon- regularity of the PiPrOx chain. Recently, it was reported that the copolymerization of iPrOx omers with R1 substituents other than isopropyl was carried out to disrupt the regularity with 2-methyl-2-oxazoline (MetOx) caused a decrease in the ability of the copolymer to of the PiPrOx chain. Recently, it was reported that the copolymerization of iPrOx with 2- crystallize when compared to PiPrOx. The LCST of the obtained copolymers changed methyl-2-oxazoline (MetOx) caused a decrease in the ability of the copolymer to crystal- significantly, and moreover, prolonged incubation of the iPrOx/MetOx copolymers at lize when compared to PiPrOx. The LCST of the obtained copolymers changed signifi- an elevated temperature caused further crystallization [28]. The other approach utilized cantly, and moreover, prolonged incubation of the iPrOx/MetOx copolymers at an ele- the copolymerization of iPrOx with its structural isomer 2-n-propyl-2-oxazoline (nPrOx), vated temperature caused further crystallization [28]. The other approach utilized the co- which led to a decrease in the ability of the copolymer to crystallize and a slight decrease polymerization of iPrOx with its structural isomer 2-n-propyl-2-oxazoline (nPrOx), which in LCST (only of a few ◦C) when compared to PiPrOx. Unfortunately, the prolonged heatingled to a decrease of iPrOx/nPrOx in the ability also of caused the copolymer further crystallization,to crystallize and similar a slight to decrease the case in of LCST the iPrOx/MetOx(only of a few copolymers°C) when compared [25]. Attempts to PiPrOx. to introduce Unfortunately, ethylene the imine prolonged (EI) units heating within of theiPrOx/nPrOx iPrOx-copolymer also caused chain further led to thecrystallization, suppression similar of crystallization to the case even of the upon iPrOx/MetOx prolonged annealing,copolymers which [25]. Attempts was attributed to introduce to the ethylene increased imine elasticity (EI) units of the within chains the induced iPrOx-copol- by EI segments,ymer chain but led simultaneously, to the suppression a significant of crystallization change in the even LCST upon was prolonged observed [ 28 annealing,]. which was attributed to the increased elasticity of the chains induced by EI segments, but Based on these studies, it seems that by controlling only the R1 substituent of the comonomer,simultaneously, it is a not significant possible change to disorder in the the LCST chain was regularity observed of [28] iPrOx-based. copolymers and, consequently,Based on these to studies, suppress it itsseems ability that to by crystallize, controlling with only simultaneous the R1 substituent control ofof thethe LCSTcomonomer, at the desired it is not level. possible to disorder the chain regularity of iPrOx-based copolymers and, Inconsequently, this work, we to aimedsuppress to synthesize its ability to copolymers crystallize, of with iPrOx simultaneous with 2,4-disubstituted-2- control of the oxazolineLCST at the and desired to check level. whether, by an appropriate selection of R1 and R2 substituents of theIn comonomer, this work, we it isaimed possible to synthesize to suppress copolymers the crystallization of iPrOx with of the 2,4 copolymer-disubstituted while-2- simultaneouslyoxazoline and to maintaining check whether, the LCSTby an appropriate close to the selection human body of R1temperature. and R2 substituents For this of reason,the comonomer, we carried it outis possible the copolymerization to suppress the of crystallization iPrOx with 2-ethyl-4-methyl-2-oxazoline of the copolymer while sim- (EtMetOx).ultaneously We maintaining expected thatthe LCST the methyl close to substituent the human R 2bodypresent temperature. within the For main this chain reason, of thewe iPrOx carried copolymer out the copolymerizationwould disorder the of chain-specific iPrOx with arrangement, 2-ethyl-4-methyl which-2-oxazoline is responsible (Et- forMetOx). the ability We expected of the copolymer that the methyl to crystallize. substituent At R the2 present same within time, we the expected main chain that of thethe ethyliPrOx substituent copolymer Rwould1 would disorder ensure the an chain appropriate-specific hydrophilic/lipophilic arrangement, which is balance responsible and anfor LCSTthe ability near of the the physiological copolymer to temperature. crystallize. At The the cytotoxicity same time, ofwe the expected copolymer that the must ethyl be testedsubstituent to check R1 would its bioapplicability. ensure an appropriate The synthesized hydrophilic/lipophilic polymers were foundbalance to and be nontoxican LCST tonear fibroblasts the physiological at the concentrations temperature. requiredThe cytotoxicity for biomedical of the copolymer tests. Such must noncrystalline be tested to check its bioapplicability. The synthesized polymers were found to be nontoxic to

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fibroblasts at the concentrations required for biomedical tests. Such noncrystalline copol- copolymersymers of 2-oxazolines of 2-oxazolines exhibiting exhibiting a phase a phase transition transition around around body body temperature temperature have have not been obtained previously and could bebe interesting candidatescandidates forfor thethe designdesign ofof polymericpolymeric nanosystems for a wide range ofof biomedicalbiomedical applications.applications.

2. Results and DiscussionDiscussion As claimed,claimed, we we aimed aimed to to introduce introduce an an alkyl alkyl substituent substituent into into the main the main chain chain of the iPrOxof the copolymeriPrOx copolymer to disorder to disorder its regularity its regularity and speciﬁc and arrangement,specific arrangement, which should which subsequently should sub- decreasesequently its decrease ability to its crystallize. ability to Forcrystallize. this reason, For this copolymerization reason, copolymerization of iPrOx with of 2,4- iPrOx or 2,5-disubstituted-2-oxazolinewith 2,4- or 2,5-disubstituted is-2- necessary.oxazoline CROPis necessary. of 2-oxazolines CROP of substituted2-oxazolines with substituted R2 leads towith a higher R2 leads conversion to a higher of conversion the monomer of the when monomer compared when to compared 2-oxazolines to 2 with-oxazolines R3. Usually, with 2-oxazolinesR3. Usually, 2 with-oxazolines R2 substituted with R2 substituted by methyl [by29 –methyl31] or [29 ethyl–31] [17 or,32 ethyl–35] [17,32 groups–35] are groups used, butare used, the polymerization but the polymerization of 2-oxazolines of 2-oxazolines with –C(O)OCH with –C(O)OCH3 or phenyl3 or [ 35phenyl]R2 substituents [35] R2 sub- isstituents also known. is also As, known. at the As, same at the time, same we time, aimed we to aimed assure to the assure LCST the at aLCST temperature at a tempera- close toture the close physiological to the physiological value, the rolevalue, of thethe Rrole1 substituent of the R1 substituent of the comonomer of the comonomer is also crucial. is Toalso provide crucial. an To appropriate provide an hydrophilic/lipophilicappropriate hydrophilic/lipophilic balance of thebalance copolymer, of the copolymer, the proper lengththe proper of the length R1 alkyl of substituent the R1 alkyl must substituent be selected must and be the selected amount ofand the the comonomer amount of must the becomonomer controlled. must be controlled. For this this reason, reason, copolymers copolymers of of 2-ethyl-4-methyl-2-oxazoline 2-ethyl-4-methyl-2-oxazoline (EtMetOx) (EtMetOx) and iPrOx iPrOx with different contentscontents of EtMetOx were synthesized via CROP. TheThe theoreticaltheoretical degreedegree ofof polymerization (DP)(DP) waswas assumedassumed toto bebe 100 and the copolymer composition waswas assumed with aa DPDP ofof EtMetOxEtMetOx of of 10 10 or or 50. 50. The The chemical chemical structure structure of of the the copolymers copolymers is presentedis presented in Figurein Figure2. 2.

Figure 2.2. Structure of the copolymers ofof 2-ethyl-4-methyl-2-oxazoline2-ethyl-4-methyl-2-oxazoline (EtMetOx)(EtMetOx) andand 2-isopropyl-2-2-isopropyl- oxazoline2-oxazoline (iPrOx). (iPrOx).

Based on the monomer consumption rate, copolymers of the random structure werewere obtained. The DP of the macromolecules was calculated on the basisbasis ofof thethe conversionconversion ofof comonomers andand NMRNMR data,data, andand thethe copolymerscopolymers werewere denoteddenoted as as P(EtMetOx P(EtMetOx1010-iPrOx-iPrOx9090) and P(EtMetOx P(EtMetOx5050-iPrOx-iPrOx5050). ).Good Good agreement agreement of the of molar the molar mass mass with withthe theoretical the theoretical value −1 −1 valuewas achieved, was achieved, as obtained as obtained from GPC from-MALLS GPC-MALLS (Mn = 13 (M,000n = g 13,000 mol , gÐmol = 1.14 ,andÐ = M 1.14n = 8500 and −1 −1 Mg moln = 8500, Ð = g 1.34, mol respectively), Ð = 1.34, respectively) (Supplementary (Supplementary Figures S1 and Figures S2). A S1 panel and of S2). properties A panel of(crystallization properties (crystallization in aqueous solution in aqueous and solutionin the solid and-state) in the was solid-state) analyzed was for analyzed the first forco- thepolymer ﬁrst copolymer P(EtMetOx P(EtMetOx10-iPrOx90) 10due-iPrOx to its90 transition) due to its temperature transition temperature being close beingto the closehuman to thebody human temperature body temperature (see below). (see The below).second Thecopolymer second P(EtMetOx copolymer50 P(EtMetOx-iPrOx50) was50-iPrOx used for50) wascytotoxicity used for studies cytotoxicity as its studiestransition as itstemperature transition temperatureensured the ensuredsolubility the of solubility the polymer of the in polymerthe medium in the at mediumculture conditions at culture. conditions.

2.1. Properties in the Aqueous Solution

ThermosensitivityThermosensitivity studies by turbidimetry revealed that thethe LCSTLCST ofof P(EtMetOxP(EtMetOx1010- ◦ iPrOx90)) was was equal equal to to 38 38 °C,C, which which is the is the same same as that as thatof PiPrOx of PiPrOx [14] (Fig [14]ure (Figure 3a). The3a). phase The phasetransition transition of the ofcopolymer the copolymer was completely was completely reversible. reversible. When When the polymer the polymer solution solution was was heated above the cloud point temperature (T ), given as 50% transmittance at the heated above the cloud point temperature (TCP), givenCP as 50% transmittance at the heating heatingcycle, and cycle, then and cooled then cooledto room to temperature, room temperature, practically practically no hysteresis no hysteresis of transition of transition was wasobserved observed (Figure (Figure 3b).3 Anb). important An important factor factor influencing inﬂuencing the biomedical the biomedical applicability applicability of pol- of polymersymers is is the the presence presence of of various various salts salts in the in solution the sol andution their and effect their on the effect thermosensi- on the tivity of the polymers [36,37]. Figure3c shows the cloud points of the copolymer recorded

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thermosensitivity of the polymers [36,37]. Figure 3c shows the cloud points of the copol- in the presence of increasing amounts of sodium chloride. A slight salting-out effect is ymer recorded in the presence of increasing amounts of sodium chloride. A slight salting- observed as the presence of sodium chloride leads to a natural partial dehydration of the out effect is observed as the presence of sodium chloride leads to a natural partial dehy- polymer; however, the change in TCP is rather subtle, similar to the case of PiPrOx [38]. In dration of the polymer; however, the change in TCP is rather subtle, similar to the case of conclusion, when short-term heating conditions are applied, P(EtMetOx10-iPrOx90) exhibits PiPrOxa thermoresponsive [38]. In conclusion, behavior when comparable short-term to heating that of PiPrOx.conditions are applied, P(EtMetOx10- iPrOx90) exhibits a thermoresponsive behavior comparable to that of PiPrOx.

(a) (b)

CP 10 90 Figure 3. ((aa)) Plot Plot of of T TCP asas a afunction function of of the the P(EtMetOx P(EtMetOx10-iPrOx-iPrOx90) concentration) concentration,, (b (b) )transmittance transmittance-temperature-temperature dependence dependence of the aqueous solution of the copolymer (c = 5 g L−−11), (c) plot of TCP as a function of the NaCl concentration (the copolymer of the aqueous solution of the copolymer (c = 5 g L ), (c) plot of TCP as a function of the NaCl concentration (the copolymer concentration in water is 5 g L−1) and (d) transmittance-temperature dependence of the aqueous solution of the copolymer concentration in water is 5 g L−1) and (d) transmittance-temperature dependence of the aqueous solution of the copolymer (c = 5 g L−1) kept for 12 h at 50 °C before cooling. (c = 5 g L−1) kept for 12 h at 50 ◦C before cooling.

Incubation of P(EtMetOx10-iPrOx90) in water at a temperature above TCP (50 °C)◦ for 12 Incubation of P(EtMetOx10-iPrOx90) in water at a temperature above TCP (50 C) for h12 led h led to a to full a full return return to totransparency transparency of of the the solutions solutions when when the the temperature temperature was was decreased decreased (Fig(Figureure 3d).d). Macroscopically, Macroscopically, this this fact fact indicates indicates a alack lack of of crystallization crystallization in inthe the solution, solution, in contrastin contrast to PiPrOx, to PiPrOx, where where the solution the solution after after incubation incubation under under similar similar conditions conditions and cool- and ingcooling remained remained cloudy cloudy and andthe theseparated separated precipitate precipitate was was crystalline crystalline [15,39] [15,39. However,]. However, a hysteresisa hysteresis of ofthe the phase phase transition transition of ofapproximately approximately 2 °C 2 ◦wasC was found found for forthe thecopolymer copolymer in- CP cubatedincubated for for a prolonged a prolonged time time at attemperature temperature above above T T.CP . Briefly,Brieﬂy, P(EtMetOxP(EtMetOx1010--iPrOxiPrOx9090),), similar similar to to PiPrOx, PiPrOx, exhibits exhibits a a LCST LCST near the physiolog- physiologi- icalcal temperaturetemperature and it is slightly dependent on the concentration and and salts. salts. Importantly, Importantly, thethe copolymer, copolymer, in in contrast contrast to to PiPrOx, PiPrOx, does does not not crystallize crystallize in in water water when when long long-term-term heating heating cconditionsonditions are are applied. applied.

2.2.2.2. Properties Properties in in the the Solid Solid-State-State

TheThe thermal thermal and and crystalline crystalline properties of P(EtMetOx1010--iPrOxiPrOx90)) in in the the solid solid-state-state were were studiedstudied by by DSC DSC and and WAXS. WAXS. ItIt can can be be seen seen that that neither neither crystallization crystallization nor nor melting melting is is observed observed during during the the standar standardd ◦ ◦ (10(10 °C/min)C/min) or or even even slow slow (2.5 (2.5 °C/min)C/min) DSC DSC measurements, measurements, indicating indicating the the low low tendency tendency of

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of this copolymer to crystallize (Figure4a). Moreover, after thermal treatment (annealing this copolymer to crystallize (Figure 4a). Moreover, after thermal treatment (annealing of of the copolymer at 180 ◦C for 1 h, and then cooling to room temperature at a rate of the copolymer at 180 °C for 1 h, and then cooling to room temperature at a rate of 10 10 ◦C/min), the lack of exo- or endothermic peaks can be seen in the DSC trace. Such °C/min), the lack of exo- or endothermic peaks can be seen in the DSC trace. Such behavior behavior is different from that of PiPrOx, where the substantial effects from crystallization is different from that of PiPrOx, where the substantial effects from crystallization at ~150 at ~150 ◦C and melting at ~200 ◦C were observed in the DSC traces [16,27]. °C and melting at ~200 °C were observed in the DSC traces [16,27].

(a) (b)

FigureFigure 4. (a) DSC traces and (b) X-rayX-ray diffraction curvecurve ofof P(EtMetOxP(EtMetOx1010--iPrOxiPrOx90).).

TheThe glass transitiontransition temperaturetemperature (T (Tg)g) of of the the copolymer, copolymer, determined determined from from measurement measure- mentafter theafter first the run first and run quenching and quenching with liquid with nitrogen,liquid nitrogen, was equal was to equal 68 ◦C, to which 68 °C, is thewhich same is theasthat same for a PiPrOxs that for [27 PiPrOx,39] (Supplementary [27,39] (Supplementary Figure S3). Additionally,Figure S3). Additionally, no diffraction no peaks diffrac- can tionbe seen peaks in thecan WAXS be seen curve in the for WAXS P(EtMetOx curve10 -iPrOxfor P(EtMetOx90) (Figure10-4iPrOxb), confirming90) (Figure the 4b conclusion), confirm- ingdrawn the fromconclusion the DSC drawn that thefrom amorphous the DSC that phase the is amorphous predominant, phase even is after predominant, 1 h of annealing even ◦ ◦ afterof the 1 hour copolymer. of annealing In contrast, of the twocopolymer. characteristic In contrast, diffraction two characteristic peaks at 2θ = diffraction 7.84 and 18.08peaks atcould 2θ = be7.84° seen and in the18.08° WAXS could curve be seen for PiPrOx in the WAXS [39]. Additionally, curve for PiPrOx we checked [39]. Additionally, whether the ◦ wevery checked prolonged whether annealing the very of theprolonged copolymer annealing (for 12 h)of atthe 180 copolymerC and further (for 12 slowh) at cooling180 °C andwould further enhance slow its cooling ability wouldto crystallize. enhance While its ability no diffraction to crystallize. peaks While could nobe seen diffraction in the peaksWAXS could curve be (Supplementary seen in the WAXS Figure curve S4a), (Supplementary small exo- and endothermicFigure S4a), small peaks exo appeared- and en- in ◦ ◦ dothermicthe DSC trace, peaks with appeared maxima in at the 155 DSCC and trace, 188 withC, respectively maxima at (Supplementary155 °C and 188 Figure°C, respec- S4b). tivelyThese ( effectsSupplementary exhibit very Figure low energyS4b). These (∆H ~0.5effects J/g); exhibit nevertheless, very low such energy behavior (H ~0.5 indicates J/g); nevertheless,that it is not possiblesuch behavior to completely indicates neglect that it theis not ability possible of seemingly to completely amorphous neglect polymers the abil- ityto crystallize.of seemingly To amorphous conclude, inpolymers the solid-state, to crystallize. P(EtMetOx To conclude,10-iPrOx 90in) the exhibits solid- astate, definitely P(Et- reduced ability to crystallize when compared to PiPrOx, whilst maintaining a T similar to MetOx10-iPrOx90) exhibits a definitely reduced ability to crystallize when comparedg to that of PiPrOx. PiPrOx, whilst maintaining a Tg similar to that of PiPrOx.

2.3. The Role of the R2 Methyl Substituent 2.3. The Role of the R2 Methyl Substituent We assume that the reduced ability of P(EtMetOx10-iPrOx90) to crystallize compared to We assume that the reduced ability of P(EtMetOx10-iPrOx90) to crystallize compared PiPrOx results from the presence of the methyl R2 substituent distributed within the main to PiPrOx results from the presence of the methyl R2 substituent distributed within the chain, while maintaining the LCST of the copolymer near physiological temperature results main chain, while maintaining the LCST of the copolymer near physiological temperature from the proper choice of an R1 (ethyl) substituent of the comonomer and the control of the results from the proper choice of an R1 (ethyl) substituent of the comonomer and the con- amount of 2,4-disubstituted-2-oxazoline. The appropriate selection of R1 and R2 inﬂuences trolthe of thermosensitivity the amount of 2,4 and-disubstituted ability to crystallize,-2-oxazoline. clearly The appropriate reﬂecting the selection structure–property of R1 and R2 influencesrelationships the ofthermosensitivity poly(2-oxazoline)s. and ability to crystallize, clearly reflecting the structure– property relationships of poly(2-oxazoline)s. To verify the key role of the R2 substituent in decreasing the ability of P(EtMetOx10- To verify the key role of the R2 substituent in decreasing the ability of P(EtMetOx10- iPrOx90) to crystallize, we obtained the copolymer of iPrOx with 2-ethyl-2-oxazoline (EtOx) iPrOxof a similar90) to crystallize, composition, we and obtained we compared the copolymer the thermal of iPrOx and with crystalline 2-ethyl properties-2-oxazoline of (EtOx) of a similar composition, and we compared the thermal and crystalline properties copolymers with and without R2 methyl substituents within the main chain. The chemical ofstructure copolymers of the with copolymer and without is presented R2 methyl in Figuresubstituents5. within the main chain. The chemical structure of the copolymer is presented in Figure 5.

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Figure 5. Structure of the copolymer of 2-ethyl-2-oxazoline (EtOx) and iPrOx. FigureFigure 5. 5. StructureStructure of of the the copolymer copolymer of of 2 2-ethyl-2-oxazoline-ethyl-2-oxazoline ( (EtOx)EtOx) and iPrOx iPrOx..

1 TheThe characterizationcharacterization of of P(EtOx P(EtOx1414-iPrOx-iPrOx8686) by H NMR, SEC SEC-MALLS-MALLS and turbidimetry The characterization of P(EtOx14-iPrOx86) by 1H NMR, SEC-MALLS and turbidimetry isis providedprovided inin thethe SupplementarySupplementary FigureFigure S5.S5. is provided in the Supplementary Figure S5. ◦ InIn thethe DSCDSC tracetrace of of P(EtOx P(EtOx1414-iPrOx-iPrOx8686)) recordedrecorded atat 2.52.5 C/min,°C/min, aa smallsmall endothermicendothermic In the DSC trace of P(EtOx14-iPrOx86) recorded at 2.5 °C/min, a small endothermic peakpeak cancan be seen at ~185 °◦C,C, in in which which enthalpy enthalpy significantly signiﬁcantly increased increased to to~3 ~3 J/g J/g when when the peak can be seen at ~185 °C, in which enthalpy significantly increased to ~3 J/g when the thesample sample was was thermally thermally treated treated before before measurement measurement (annealing (annealing at 180 at ° 180C for◦C 1 for h and 1 h andthen sample was thermally treated before measurement (annealing at 180 °C for 1 h and then thencooling cooling to room to room temperature temperature at a rate at aof rate 10 ° ofC/min) 10 ◦C/min) (Figure (Figure 6a). Additionally,6a). Additionally, the diffrac- the cooling to room temperature◦ at a rate of 10 °C/min) (Figure 6a). Additionally, the diffrac- diffractiontion peak at peak 2θ ~8° at 2canθ ~8 be canseen be in seen the WAXS in the WAXScurve of curve thermally of thermally treated treatedP(EtOx P(EtOx14-iPrOx1486-) tion peak at 2θ ~8° can be seen in the WAXS curve of thermally treated P(EtOx14-iPrOx86) iPrOx(Figure86) 6 (Figureb). 6b). (Figure 6b).

(a) (b) (a) (b) Figure 6. (a) DSC traces and (b) X-ray diffraction curve of P(EtOx14-iPrOx86). Figure 6. ((a) DSCDSC tracestraces andand ((b)) X-rayX-ray diffractiondiffraction curvecurve ofof P(EtOxP(EtOx1414--iPrOxiPrOx86).).

Additionally, for P(EtOx14-iPrOx86) annealed for a much longer time (12 h) at 180 ◦°C, Additionally,Additionally, for P(EtOx14--iPrOxiPrOx8686) )annealed annealed for for a a much much longer longer time time (12 (12 h) h) at at 180 180 °C,C, a pronounced endothermic peak at ~185 ◦°C (H ~13 J/g) can be seen in the DSC trace aa pronounced pronounced endothermic peak peak at ~185 °CC( (∆HH ~13 J/g) J/g) can can be be seen seen in in the the DSC trace (Supplementary Figure S6a), together with a significant diffraction peak at 2θ◦ ~8° (Sup- ((SupplementarySupplementary Figure S6a),S6a), togethertogether with with a a significant significant diffraction diffraction peak peak at at 2θ 2θ~8 ~8°(Supple- (Sup- plementary Figure S6b), indicating the considerable ability of this copolymer to crystal- plementarymentary Figure Figure S6b), S6b indicating), indicating the the considerable considerable ability ability of thisof this copolymer copolymer to crystallize.to crystallize. This finding confirms that the methyl R2 substituent strongly influences the disorder lize.This This finding finding confirms confirms that thethat methyl the methyl R2 substituent R2 substituent strongly strongly influences influences the disorder the disorder of the of the chain-specific arrangement of iPrOx-based copolymers, thus inhibiting crystalliza- ofchain-specific the chain-specific arrangement arrangement of iPrOx-based of iPrOx-based copolymers, copolymers, thus inhibiting thus inhibiting crystallization. crystallization. tion. 2.4. Cytotoxicity 2.4. Cytotoxicity 2.4. CytotoxicityThe MTT test, a standard laboratory colorimetric assay for the measurement of cellular growth,The was MTT used test, to a evaluate standard the laboratory cytotoxicity colorimetric of the copolymer. assay for P(EtMetOx the measurement50-iPrOx of50 )cellu- was The MTT test, a standard laboratory◦ colorimetric assay for the measurement of cellu- usedlar growth, for the was test dueused to to its evaluate LCST of the 41 cytotoxicityC, which means of the that copolymer. during cell P(EtMetOx culture carried50-iPrOx out50) lar growth, was used to evaluate the cytotoxicity of the copolymer. P(EtMetOx50-iPrOx50) atwas 37 used◦C, it for is the fully test soluble due to in its DMEM LCST of (Supplementary 41 °C, which means Figure that S2c). during In thiscell study,culture human carried was used for the test due to its LCST of 41 °C, which means that during cell culture carried fibroblastsout at 37 °C, were it is selected fully soluble as model in DMEM cells. Human (Supplementary fibroblasts Figure are commonly S2c). In this used study, to verify human the out at 37 °C, it is fully soluble in DMEM (Supplementary Figure S2c). In this study, human cytotoxicityfibroblasts were of polymers, selected andas model we have cells. shown Human the lackfibroblasts of toxicity are commonly of (co)poly(2-oxazoline)s used to verify fibroblasts were selected as model cells. Human fibroblasts are commonly used to verify towardsthe cytotoxicity fibroblasts of polymers, in our previous and we studies have shown [12,13 ].the The lack concentrations of toxicity of of(co)poly(2 the copolymer-oxazo- the cytotoxicity of polymers, and we have shown the lack of toxicity of (co)poly(2-oxazo- wereline)s as towards follows: fibroblasts 0.001, 0.01, in 0.1, our 1 previous and 10 mg/mL. studies [12,13] The results. The areconcentrations shown in Figure of the7. copol- line)s towards fibroblasts in our previous studies [12,13]. The concentrations of the copolymer were as follows: 0.001, 0.01, 0.1, 1 and 10 mg/mL. The results are shown in Figure 7. ymer were as follows: 0.001, 0.01, 0.1, 1 and 10 mg/mL. The results are shown in Figure 7.

Int. J. Mol. Sci. 2021, 22, 2221 7 of 11 Int. J. Mol. Sci. 2021, 22, 2221 7 of 11

FigureFigure 7.7. Cytotoxicity assayassay ofof P(EtMetOxP(EtMetOx5050-iPrOx-iPrOx5050) at increasing concentrationsconcentrations (given(given inin mg/mL).mg/mL). TheThe assayassay waswas performedperformed withwith ﬁbroblasts.fibroblasts. The results are shown as a percentagepercentage of thethe control,control, wherewhere untreateduntreated cellscells constitutedconstituted 100%.100%.

P(EtMetOxP(EtMetOx5050-iPrOx-iPrOx5050)) is is nontox nontoxicic to fibroblasts ﬁbroblasts inin aa wide rangerange ofof concentrations.concentrations. TheThe percentagepercentage ofof ﬁbroblastfibroblast viabilityviability waswas atat leastleast 70%,70%, relativerelative toto thethe control,control, exceptexcept forfor thethe highesthighest concentrationconcentration (10(10 mg/mL)mg/mL) at 72 h. The obtained resultsresults indicateindicate thatthat synthesizedsynthesized polyoxazolinespolyoxazolines maymay bebe consideredconsidered forfor futurefuture applicationsapplications inin biomedicine.biomedicine.

3.3. Materials andand MethodsMethods 3.1. Materials 3.1. Materials Isobutyronitrile (99.6%, Aldrich, Steinheim, Germany), 2-aminoethanol (99%, Aldrich, Isobutyronitrile (99.6%, Aldrich, Steinheim, Germany), 2-aminoethanol (99%, Al- Steinheim, Germany), cadmium acetate (>98%, Fluka, Steinheim, Germany), D,L-Alaninol drich, Steinheim, Germany), cadmium acetate (>98%, Fluka, Steinheim, Germany), D,L- (98%, Aldrich, Steinheim, Germany), propionitrile (>99%, Fluka, Steinheim, Germany) Alaninol (98%, Aldrich, Steinheim, Germany), propionitrile (>99%, Fluka, Steinheim, Ger- and methyl 4-nitrobenzenesulfonate (99%, Aldrich, Steinheim, Germany) were used as many) and methyl 4-nitrobenzenesulfonate (99%, Aldrich, Steinheim, Germany) were received. 2-Ethyl-4-methyl-2-oxazoline (EtMetOx) was synthesized according to Schu- bertused [ 17as]. received. 2-Isopropyl-2-oxazoline 2-Ethyl-4-methyl (iPrOx)-2-oxazoline was synthesized(EtMetOx) was according synthesized to Witte according and Seel- to igerSchubert [40]. Raw[17]. EtMetOx,2-Isopropyl iPrOx-2-oxazoline and EtOx (iPrOx) (99%, Aldrich,was synthesized Steinheim, according Germany) to were Witte dried and Seeliger [40]. Raw EtMetOx, iPrOx and EtOx (99%, Aldrich, Steinheim, Germany) were over KOH, distilled, dried over CaH2 and distilled again. Acetonitrile (for HPLC, POCH, dried over KOH, distilled, dried over CaH2 and distilled again. Acetonitrile (for HPLC, Gliwice, Poland) was dried over CaH2 and distilled under a dry argon atmosphere. Hu- manPOCH, dermal Gliwice, fibroblasts Poland) werewas dried derived over from CaH the2 and Tissue distilled Bank under at the a dry Dr. argon Stanislaw atmosphere. Sakiel CenterHuman for dermal Burns fibroblasts Treatment were in Siemianowice derived from Slaskie. the Tissue DMEM Bank growth at the Dr. medium Stanislaw (Sterile Sakiel A, Corning,Center for NY, Burns USA) Treatment was used in with Siemianowice 10% FBS (Mediatech, Slaskie. DMEM Corning, growth NY, medium USA) and (Sterile antibi- A, otic/antimycoticCorning, NY, USA) (Gentamicin/Amphotercin was used with 10% FBS B, (Mediatech, Gibco, Fisher Corning, Scientific, NY Göteborg,, USA) and Sweden). antibi- (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumotic/antimycotic (Gentamicin/Amphotercin B, Gibco, bromide) Fisher (MTT) Scientific, (CyQUANT Göteborg,TM MTT Swe- Cellden). Viability (3-(4,5 Assay-Dimethylthiazol Kit, Invitrogen-2- byyl) Thermo-2,5-diphenyltetrazolium Fisher Scientific, Göteborg, bromide) Sweden), (MTT) PBS (Sterile(CyQUANT Filtered,TM MTT Biowest, Cell Viability Kansas Assay City, MO, Kit, Invitrogen USA) and by DMSO Thermo (>99.9%, Fisher WAK–Chemie Scientific, Gö- Medicalteborg, SwedenGmbH,), Steinbach, PBS (Sterile Germany) Filtered, were Biowest used as, Kansas received. City, MO, USA) and DMSO (>99.9%, WAK–Chemie Medical GmbH, Steinbach, Germany) were used as received. 3.2. Synthesis of Copolymers 3.2. SynthesisThe molar of ratioCopolymers of the initial monomers concentration to the initiator concentration was 100:1.The molar The composition ratio of the ofinitial the comonomersmonomers concentration EtMetOx:iPrOx to the was initiator 10:90 and concentration 50:50. Poly- ◦ merizationswas 100:1. Thewere composition carried out at of 75 theC in comonomers acetonitrile to EtMetOx:iPrOx the full conversion was of 10:90 the monomers and 50:50. (checkedPolymerizations by gas chromatography).were carried out at Then,75 °C in water acetonitrile was added, to the the full mixture conversion was of stirred the mon- for 10omers min (checked at room temperature, by gas chromatography). the excess acetonitrile Then, water was was evaporated added, the and mixture the obtained was stirred poly- mersfor 10 were min driedat room by lyophilization.temperature, the The excess copolymers acetonitrile were was denoted evaporated as P(EtMetOx and the10 -iPrOxobtained90) andpolymers P(EtMetOx were 50dried-iPrOx by50 lyophilization.). The first copolymer The copolymers was used were for analyses, denoted whilstas P(EtMetOx the latter10- wasiPrOx used90) and for P(EtMetOx cytotoxicity50- studies.iPrOx50). Additionally, The first copolymer polymerization was used of for EtOx analyses, and iPrOx whilst was the

Int. J. Mol. Sci. 2021, 22, 2221 8 of 11

carried out with the same procedure. The composition of EtOx:iPrOx was set to 10:90. The obtained copolymer was denoted as P(EtOx14-iPrOx86) and its properties were compared to those of P(EtMetOx10-iPrOx90).

3.3. Measurements The molar mass and dispersity (Ð) of the copolymers were determined using a GPC- MALLS system with a multiangle laser light scattering detector (DAWN EOS, Wyatt Technologies, Santa Barbara, CA, USA, λ = 658 nm) and a refractive index detector (∆n- 1000 RI WGE DR Bures, Dallgow, Germany, λ = 620 nm). Measurements were carried out in DMF (with 5 mmol/L LiBr; flow rate of 1 mL/min) using PSS 100 Å, 1000 Å and 3000 Å GRAM columns. The composition of the copolymers was analyzed by 1H NMR. The spectra were recorded in CDCl3 using a Bruker Ultrashield spectrometer (Bruker, Billerica, MA, USA) operating at 600 MHz. DSC measurements were carried out using a TA-DSC Q2000 apparatus (TA Instru- ments, New Castle, DE, USA) under a nitrogen atmosphere with a flow rate of 50 mL/min. The measurements were taken in the range from 0 to 200 ◦C. The heating rate for the standard measurement was 10 ◦C/min and was equal to 2.5 ◦C/min for the so-called “slow-heating” measurement. To determine the glass transition temperature (Tg) of the copolymer, after the first run, the sample was quenched with liquid nitrogen and a second heating run was performed again from 0 to 200 ◦C. The enthalpy of melting or crystallization (∆H) was calculated as the area under the peak, limited by the baseline. The data were collected and then analyzed using Universal Analysis 2000 with Universal V4.5a software (TA Instruments, New Castle, DE, USA). Turbidimetric measurements of the aqueous solutions of copolymers were performed using a Specord 200 plus UV-Vis spectrophotometer (Analytik Jena, Jena, Germany) equipped with a programmable thermocontroller. The transmittance of the polymer solutions was monitored at a wavelength λ = 550 nm as a function of the temperature (heating and then cooling cycle) with constant stirring of the solution. Transmittance values were recorded every 1 ◦C after 60 s of temperature stabilization. The concentrations of the polymer solutions ranged from 1 to 10 g/L. The phase transition temperature (TCP) was defined as the temperature at which the transmittance of the copolymer solutions reached 50% of its initial value. The sample crystallinity was measured with a wide-angle X-ray diffractometer (WAXS) TUR-M62 (VEB TuR, Dresden, Germany) equipped with an HZG-3 goniometer (VEB TuR, Dresden, Germany) using Cu Kα radiation. Calculations of the intensities and positions of peaks were carried out using WAXSFIT software (WAXSFIT, Bielsko Biała, Poland).

3.4. Cytotoxicity Assay

The MTT cytotoxicity assay was used to estimate the cytotoxic properties of P(EtMetOx50- iPrOx50) in the treatment of human ﬁbroblasts. Cells were seeded in a 96-well tissue culture microplate at a concentration of 3300 cells per well and incubated in 100 µL of growth ◦ medium at 37 C and c(CO2) = 5% for 24 h. P(EtMetOx50-iPrOx50) was dissolved in the DMEM growth medium. Different concentrations of the polymer solution (0.001, 0.01, 0.1, 1 and 10 mg/mL) were added to the cells and the culture was carried out for 4, 8, 24, 48 and 72 h. A 12 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) stock solution was prepared in PBS. To detect formazan, a product of the redox reaction of viable cells, a rapid protocol using DMSO as a solubilizing agent was used. After a given culture time, the standard 100 µL of DMEM was replaced with DMEM without phenol red, as recommended by the MTT producer, because the presence of phenol red can affect the MTT assay results. Then, 10 µL of the 12 mM MTT stock solution was added to the well containing cells seeded in an appropriate concentration of the polymer and incubated at 37 ◦C for 4 h, followed by the removal of 85 µL of the medium. Fifty microliters of DMSO were added and pipetted up and down thoroughly for mixing. The microplate Int. J. Mol. Sci. 2021, 22, 2221 9 of 11

was incubated at 37 ◦C for 10 min. Then, the absorbance of the solution was measured at 540 nm using a Multiskan Sky Microplate Spectrophotometer (Thermo Scientiﬁc, Göteborg, Sweden). The number of cells was determined based on the calibration curve. The results are shown as a percentage of the control (cells seeded in DMEM without polymer).

4. Conclusions Herein, we have shown that by the copolymerization of iPrOx with EtMetOx, it is possible to significantly suppress the ability of the copolymer to crystallize when compared to PiPrOx, whilst maintaining the LCST and Tg similar to those of PiPrOx. The appropriate choices of R1 (ethyl) and R2 (methyl) substituents of the comonomer used for copolymerization with iPrOx were crucial for controlling the properties of the final copolymer. By the introduction of the R1 ethyl substituent of the comonomer and the control of amount of 2,4-disubstituted-2-oxazoline, the thermosensitivity could be controlled at the desired level. P(EtMetOx10-iPrOx90) exhibited a thermoresponsive behavior in water comparable to that of PiPrOx (no hysteresis of transition, LCST = 38 ◦C, a slight salting-out effect induced by the addition of NaCl). The presence of the methyl substituent R2 randomly distributed within the main chain of the copolymer disrupted the chain regularity and led to a reduced ability of the copolymer to crystallize. Unlike PiPrOx, when long-term heating conditions in water were applied (12 h at 50 ◦C), no crystallization of the copolymer was observed, as revealed by turbidimetry. Additionally, P(EtMetOx10-iPrOx90) in the solid-state exhibited a definitely reduced ability to crystallize when compared to PiPrOx, whilst maintaining a Tg similar to that of PiPrOx, as confirmed by differential scanning calorimetry and X-ray diffractometry. The copolymer was nontoxic to fibroblasts over a wide range of concentrations, which was confirmed by the calorimetric MTT assay. On the basis of the obtained results, including the limited ability to crystallize, LCST close to the physiological temperature and Tg similar to that of PiPrOx, together with its nontoxicity to fibroblasts, we can conclude that P(EtMetOx10-iPrOx90) seems to have interesting prospects for many bioapplications, e.g., in tissue engineering or controlled drug delivery, and can clearly be an alternative to the “gold standard” thermoresponsive polymers.

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/4/2221/s1. Author Contributions: All authors contributed to this study. W.W., supervision, analysis and interpretation of the results, review and editing; A.K.-B., biological studies, editing; A.S., biological studies, editing; A.K., analysis and interpretation of the results, preparation of the manuscript, review and editing; N.O.-T., concept, synthesis and characterization of copolymers, analysis of results, original draft preparation. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Centre, project 2016/21/D/ST5/01951. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data available on request. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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