Cryogels Based on Poly(2-oxazoline)s through Development of Bi- and Trifunctional Cross-Linkers Incorporating End Groups with Adjustable Stability

1,4-Bis(iodomethyl)benzene and 1,3,5-tris(iodomethyl)benzene were used as initiators for the cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazoline (EtOx) and its copolymerization with tert-butyl (3-(4,5-dihydrooxazol-2-yl)propyl)carbamate (BocOx) or methyl 3-(4,5-dihydrooxazol-2-yl)propanoate (MestOx). Kinetic studies confirmed the applicability of these initiators. Termination with suitable nucleophiles resulted in two- and three-armed cross-linkers featuring acrylate, methacrylate, piperazine-acrylamide, and piperazine-methacrylamide as polymerizable ω-end groups. Matrix-assisted laser desorption/ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed the successful attachment of the respective ω-end groups at all initiation sites for every prepared cross-linkers. Except for acrylate, each ω-end group remained stable during deprotection of BocOx containing cross-linkers. The cryogels were prepared using EtOx-based cross-linkers, as confirmed by solid-state NMR spectroscopy, scanning electron microscopy, and thermogravimetric analysis. Stability tests revealed a complete dissolution of the acrylate-containing gels at pH = 14, whereas the piperazine-acrylamide-based cryogels featured excellent hydrolytic stability.


INTRODUCTION
Cryogels represent a class of three-dimensional (3D)structured materials consisting of cross-linked polymer networks.In contrast to hydrogels, these materials are characterized by an interconnected pore structure with enhanced mechanical properties enabling mass-transport of substances. 1,2Typically, cryogels are applied for controlled drug delivery, 3 tissue engineering, 4 as bioreactors, 5 for heavy metal adsorption, 6 for separation purposes, 7 and also as 3D scaffolds for the cultivation of cells and microorganisms. 8,9ost commonly, N,N′-methylenebis(acrylamide) ( M B A A m ) , 1 0 , 1 1 e t h y l e n e g l y c o l d i m e t h a c r y l a t e (EGDMA), 1 2 , 1 3 poly(ethylene glycol) diacrylate (PEGDA), 14,15 and poly(ethylene glycol) dimethacrylate (PEGDMA) 16,17 are used as cross-linking agents for the preparation of synthetic polymer-based cryogels via free-radical cross-linking polymerization at subzero temperatures.In particular, the poly(ethylene glycol) (PEG)-based cross-linker are frequently applied when biomedical applications are targeted. 14,15,17As an example, cryogels based on poly-(ethylene glycol) methyl ether methacrylate, PEGMA, and 2-(N-succinimidylcarboxyoxy)ethyl methacrylate were prepared and functionalized with the amine-containing anticancer drug doxorubicin. 18drophilic poly(2-oxazoline)s (POx) are currently of high interest as PEG alternatives in that context. 19,20Polymerizable end groups can be easily attached due to the living nature of the cationic ring-opening polymerization (CROP), which is used to obtain POx.−26 Alternatively, the CROP can be terminated by Ntert-butyloxycarbonylpiperazine, 27−30 which has been exploited for further postpolymerization end group modifications. 27,29he termination with piperazine-based amines as nucleophiles also allows to attach radically polymerizable moieties in a direct fashion, as reported by Rueda et al., who used N-(4vinylbenzyl)piperazine as a CROP termination agent. 31he use of bi-or multifunctional CROP initiators enables access to polymers or oligomers bearing two or more radically polymerizable ω-end groups. 32Those have been applied as cross-linkers for the preparation of films, 33,34 hydrogels, 26,35,36 microbeads, 37 coatings, 38 or nanofibers 39 and remain of high interest to date.However, their potential has rarely been exploited with respect to cryogels.To the best of our knowledge, only one POx containing cryogel has been reported, 40 which is mainly based on N,N-dimethylacrylamide and only contains 14 mol % of poly(2-ethyl-2-oxazoline) diacrylate (PEtOxDA).
Cryogels prepared from solely cross-linkers are rare, although PEGDA has been applied for that purpose. 15,41It is more common to adjust the cross-linker density and cryogel properties by copolymerization with low molar mass acrylate or methacrylate monomers.However, adjustment of the crosslinker density could also be realized by the use of multifunctional cross-linkers, e.g., by introduction of oxazoline monomers to serve as radically polymerizable moieties. 42In addition, the use of bi-or multifunctional CROP initiators such as 1,4-dibromo-2-butene, 43,44 1,4-bis(bromomethyl)benzene 45 or 1,4-bis(iodomethyl)benzene (BIB) in combination with functional termination agents to yield polymerizable ω-end groups appears convenient.A comprehensive review of star-shaped POx using a variety of multifunctional initiators, including bi-and trifunctional initiators, has been published recently. 46Many multifunctional CROP initiators suffer from sterical hindrance and slow initiation, 46,47 a drawback that can be overcome by enhancing the leaving group quality at the initiator. 48It is hence surprising that the approach has not yet been investigated for multiple initiators based on benzyl halides, whose reactivity can be increased by exchanging the bromide by iodide. 49he use of low molar mass comonomers enables the introduction of moieties for the additional functionalization of cryogels.However, such groups can also be already present in a suitably designed cross-linker.As prominent examples, such as carboxylic acids and primary amines, are not tolerated by the CROP mechanism, they have to be introduced by monomers bearing suitable protection groups.Methyl 3-(4,5-dihydrooxazol-2-yl)propanoate (MestOx) 50 contains a carboxylic acid methylester, which can be functionalized directly by amidation without prior deprotection. 51[3-[(tert-Butoxycarbonyl)-amino]propyl]-4,5-dihydrooxazole (BocOx) 52 features a Bocprotected primary amino group.Once deprotected after polymerization of the monomer, BocOx provides access to subsequent functionalization with, e.g., molecules for targeting purposes 53 or dyes. 54e present the development of cryogels based on purely POx by exploring difunctional CROP initiator BIB as well as trifunctional initiator 1,3,5-tris(iodomethyl)benzene (TIB).Aiming toward cryogels with varied hydrolytic stability, polymerizable ω-end groups based on acrylate, methacrylate, acrylamide, and methacrylamide were attached via direct termination of the CROP (Scheme 1).In addition to homopolymerization of EtOx to result in hydrophilic crosslinkers, the introduction of further functional moieties was tackled by copolymerization with MestOx as well as BocOx.The concept was proven by preparation of cryogels based on PEtOx, which were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy.

EXPERIMENTAL SECTION
Detailed information concerning the materials and instruments as well as procedures describing the synthesis of initiators, monomers, and termination agents can be found in the Supporting Information.
2.1.General Procedure for Kinetic Studies.All kinetic studies were performed at an overall [M] 0 = 2 mol L −1 in acetonitrile.The ratio of [initiating moieties] to [M] 0 was kept constant at 5. This resulted in a [M] 0 /[I] 0 ratio of 10 when the bifunctional initiator BIB was used, whereas [M] 0 /[I] 0 = 15 for the trifunctional initiator TIB.In copolymerizations, 68 mol % of EtOx and 32 mol % of MestOx or BocOx, respectively, were used.Detailed amounts are provided in the Supporting Information.
A preheated flask was cooled to room temperature under argon.Subsequent to heating under vacuum refilling with argon thrice, the flask was charged with initiator, i.e., BIB or TIB, respectively, under an inert atmosphere.The predetermined amounts of monomer were added, i.e., EtOx, EtOx and MestOx or BocOx and EtOx, respectively.Subsequent to the addition of acetonitrile, the reaction mixture was heated to reflux using a heat-on system under a continuous gentle argon flow.100 μL aliquots were taken at preselected time points (5,  10, 15, 30, 45, 60, 90, 120, and 150 min, respectively) to determine the monomer conversion via gas chromatography (GC) and the molar mass distribution via size exclusion chromatography (SEC).
Scheme 1. Schematic Representation of the Synthesis Route of the POx-Based Cross-Linkers Using BIB or TIB an as Initiator a a EtOx, MestOx, and BocOx were used as monomers.Acrylic acid (A), methacrylic acid (MA), piperazine-acrylamide in the form of its trifluoroacetic acid salt (PipA), and piperazine-methacrylamide (PipMA) were used as termination agents.
The polymerization rate coefficient k p of the corresponding monomer was calculated by the postulation of a linear fit according to eqs 1 and 2.
The k p values per side chain were calculated by applying eqs 3 and 4.
The resulting values are summarized in Table 1 2.2.General Procedure for the Cross-Linker Synthesis.The cross-linker molecules were synthesized in a similar fashion as described above for the kinetic studies.Instead of sampling during the CROP, the reactions were terminated with triethylammonium acrylate, triethylammonium methacrylate, N-acryloyl-piperazinium trifluoroacetate (PipA) or N-methacryloyl-piperazine (PipMA).Detailed amounts and purification procedures for the termination agents and for each cross-linker are provided in the Supporting Information.The molecular structure of PipA was further authenticated by X-ray crystallography (see Table S1 and Figure S1).
A preheated flask was cooled to room temperature under argon.Subsequent to heating under vacuum refilling with argon thrice, the flask was charged with initiator, i.e., BIB or TIB, respectively, under an inert atmosphere.The predetermined amounts of monomer were added, i.e., EtOx, EtOx and MestOx or BocOx and EtOx, respectively.Subsequent to the addition of acetonitrile, the reaction mixture was heated to reflux using a heat-on system under a continuous gentle argon flow.Reactions times varied between 1.5 and 4 h, as optimized during the kinetic studies.Subsequently, the termination agents (1.3− 2 equiv with respect to oxazolinium chain ends) were added under an inert atmosphere.The reaction mixtures were stirred at room temperature, 50 °C or overnight.Subsequent to taking an aliquot for analysis by means of 1 H NMR spectroscopy and SEC, chloroform or dichloromethane was added.Excess amounts of termination agent and formed salts were removed by repeated washing steps with aqueous NaHCO 3 solution, brine, and deionized water.The organic phase was dried by using Na 2 SO 4 , filtered, and concentrated under reduced pressure.Some cross-linkers were additionally precipitated from diethyl ether (−20 °C).All samples were dried in vacuo and analyzed by means of 1 H and 13 C NMR spectroscopy, SEC, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
2.3.General Procedure for Deprotection of BocOx Containing Cross-Linkers.The BocOx containing cross-linkers were dissolved in dichloromethane, and trifluoroacetic acid (7−30 equiv per Boc protection group) was added.The reaction mixture was stirred at room temperature until gas evolution ceased (about 4 h).The solvent was evaporated under a reduced pressure.Purification procedures varied and are specified for each cross-linker in the Supporting Information.The amount of residual trifluoroacetate ions was determined by 19 F NMR spectroscopy using potassium fluoride as an external standard.Further characterization methods included 1 H and 13 C NMR spectroscopy as well as MALDI-TOF MS.Data are provided in the Supporting Information.
2.4.General Procedure for Cryogel Preparation.For the cryogel preparations, 5 mL polypropylene syringes were used as reaction containers.For each condition, duplicates were carried out by preparing an 8 mL monomeric stock solution and distributing 3.9 mL into two 5 mL syringes each.An aqueous monomeric solution of 0.2 mol % acryloxyethyl thiocarbamoyl rhodamine B (RhoB), the corresponding cross-linker, and K 2 S 2 O 8 was homogenized in an ultrasonic bath and was purged with argon at 0 °C for 30 min.The solution was taken up into a 5 mL syringe before addition of an aqueous N,N,N′,N′-tetramethylethylenediamine solution (80 μL of a 1.114 M solution, 0.089 mmol) through the bottom end.The syringe was bottom-capped using a syringe stopper and vortexed for 10 s.Cryo-polymerization was carried out by placing the capped syringe in a cryostat cooling bath (at −12 °C) overnight using a perforated polystyrene grid.Afterward, the syringe was removed from the cryostatic bath and thawed at room temperature for 1 h.The resulting cryogel was immersed in water to remove unreacted monomers with subsequent solvent changes for 5 days.Cryogel monoliths were cut into slices using a steel blade followed by lyophilization for at least 2 days.Slices were used for SEM imaging analysis, confocal laser scanning microscopy (CLSM), swelling experiments, and pH stability tests.Three or four slices were ground to a fine powder for analysis by means of ssNMR spectroscopy and thermogravimetric analysis (TGA).The exact experimental details are summarized in Tables S2 and S3 in the Supporting Information.

RESULTS AND DISCUSSION
The goal of this work was the synthesis of two-and threearmed cross-linkers based on oligomeric 2-oxazolines (Scheme 1) for the preparation of cryogels.The development included the exploration of bi-and trifunctional CROP initiators as well as the design of suitable termination agents to introduce radically polymerizable ω-end groups in a direct fashion.
3.1.Kinetic Studies.CROP initiators comprising multiple initiation sites represented the basis for the development of the POx-based cross-linkers.Benzyl halides have long been known as suitable CROP initiators. 55,56Particularly, a range of benzyl bromides is commercially available.However, some of them tend to cause slow initiation, which can lead to multimodal molar mass distributions for multifunctional initiators. 57−60 Therefore, two initiators based on iodomethylbenzene derivatives were synthesized via Finkelstein reactions in order to obtain symmetrical two-and three-armed cross-linkers (Scheme 1).The corresponding bi-and trifunctional initiators 1,4-bis-(iodomethyl)benzene (BIB) and 1,3,5-tris(iodomethyl)benzene (TIB), respectively, were obtained according to the literature. 61,62However, TIB has not been used as an initiator for the CROP of 2-alkyl-2-oxazolines to the best of our knowledge.
To explore the feasibility of these compounds as CROP initiators, kinetic studies using EtOx as a monomer were conducted in acetonitrile at an initial monomer concentration [M] 0 of 2 mol L −1 under reflux conditions to minimize chain transfer reactions during the CROP.The latter would result in the formation of proton-initiated species and, in consequence, would lead to impurities with single instead of multiple ω-end groups. 63n view of the applications as cross-linkers, the degree of polymerization per arm was set to five.This resulted in a total [M] 0 /[I] 0 of 10 for the bifunctional initiator BIB and in a total [M] 0 /[I] 0 of 15 for the trifunctional initiator TIB.
The semi-logarithmic kinetic plots ln([M] 0 /[M] t ) versus reaction time (Figure 1) increased in a linear fashion for both initiators.This is characteristic of a pseudo-first-order behavior with a rapid and efficient initiation.The different [M] 0 /[I] 0 ratio resulted in apparent k p values that deviated when comparing BIB and TIB (compare entries for B-EtOx and T-EtOx, respectively, in Table 1).However, [M] 0 /[I] 0 per initiation site was kept constant as five.In consequence, the k p,arm values were very similar, demonstrating an efficient initiation at each site for the bifunctional initiator BIB as well as for the trifunctional initiator TIB.The molar mass (M n ) increased in a linear fashion when plotted against monomer conversion for both initiators, indicating that the molar mass can be well controlled.The monomodal molar mass distributions with narrow dispersity values (Đ < 1.2) confirmed the efficient and simultaneous initiation at all sites (Figures S2 and S3 in the Supporting Information).
We next investigated copolymerization reactions of EtOx with monomers featuring functional moieties.The methylester containing monomer MestOx and the Boc-protected primary amine containing BocOx were synthesized according to literature procedures. 50,52The fraction of functional monomer was set as ≈30% for both initiators.Therefore, the molar ratio of EtOx to functional comonomer was adjusted to 7:3 for bifunctional initiator BIB, whereas the ratio was 10:5 for trifunctional initiator TIB.Thereby, the targeted DP value of five per arm was maintained.
The copolymerization of EtOx with MestOx was of pseudofirst order, as indicated by the kinetic studies.The linear evolution of the molar mass with conversion was retained for both initiators, as were the monomodal molar mass distributions (Đ < 1.2, Figures S4 and S5 in the Supporting Information).The semi-logarithmic plots (Figure 1, second row) showed that the polymerization rates of EtOx and MestOx were very different from each other, which is wellknown from the literature. 50As evident from the slope and the k p,arm values (Table 1), EtOx was incorporated twice as fast as MestOx.Whereas these observations applied to both initiators, individual k p,arm values differed from each other.The polymerization rate coefficient of EtOx was similar to that of the homopolymerization when using the bifunctional initiator BIB (compare entries for B-EtOx vs B-MestOx in Table 1).However, CROP using the trifunctional initiator TIB was accelerated by a factor of 2 when MestOx was used as a comonomer (compare entries for T-EtOx vs T-MestOx in Table 1).This acceleration of the propagation in case of using TIB could be related to the activation of the chain ends and/or stabilization of the transition state by the MestOx residues in the immediate vicinity as reported in the literature. 50,64As a result, potentially two MestOx units could stabilize the oxazolinium species.However, due to the symmetrical structure of BIB, these corresponding carbonyl groups are not in close vicinity, which may cause the difference.
In contrast, the use of BocOx as a functional comonomer did not significantly affect the propagation rate of EtOx (Table 1).The semi-logarithmic plot (Figure 1, third row) revealed a similar behavior of both monomers with almost overlapping linear regressions.The CROP tended to be slightly decelerated when the trifunctional initiator TIB was used.The M n against conversion plot featured linear behavior as soon as conversions of >50% were reached.Deviations from linearity at lower conversions are due to overlapping monomer signals in the SEC elugrams (see Figures S6 and S7 in the Supporting Information).

Cross-Linker Synthesis by Termination of the CROP.
As all kinetic studies hinted toward the presence of a living CROP, the next step included the termination of the oxazolinium chain ends with nucleophiles bearing radically polymerizable moieties.This direct termination approach avoided the need of further modification steps subsequent to polymerization within the cross-linker synthesis route.Acrylate, methacrylate, acrylamide as well as methacrylamidebased cross-linkers were designed to cover a range of common radically polymerizable moieties.−25 Acrylamides and methacrylamides were introduced through piperazine derivatives as termination agents.For this purpose, N-acryloyl-piperazinium trifluoroacetate (PipA) and N-methacryloyl-piperazine (PipMA) were synthesized according to literature procedures. 65,66For the synthesis of PipA, N-Boc-piperazine was treated with acryloyl chloride to yield the Boc-protected amide.After deprotection by the addition of TFA and recrystallization in diethyl ether, the pure product was obtained in an overall yield of 75% and analyzed by 1 H NMR spectroscopy and X-ray crystallography (see Table S1 and Figure S1 in the Supporting Information).PipMA was obtained as a yellow oil in rather low yields (25%) by the reaction of piperazine with methacrylic anhydride. 65he use of these four termination agents, the two initiators, and the monomer systems EtOx, EtOx/MestOx, and EtOx/ BocOx resulted in 20 end-functionalized POx as listed in Table 2.The BocOx containing POx was subsequently deprotected to yield cross-linkers featuring amino moieties (AmOx).The cross-linkers were analyzed by means of SEC, 1 H NMR spectroscopy as well as MALDI-TOF MS.The resulting characterization data are summarized in Table 3.
All SEC elugrams revealed the presence of monomodal molar mass distributions indicating a uniform chain propagation at all initiating sites using both initiators (Figures S8−S27 the Supporting Information).
The presence of the desired ω-end groups was confirmed by 1 H NMR spectroscopy.Vinylic proton signals were evident in the spectra of all cross-linkers (Figures S8−S27 in the Supporting Information).Integral values of these were used to estimate the degree of functionalization (DF) with the ωend group by comparison with signal integrals derived from the benzylic methylene protons of the initiator.−26 PipA and PipMA have, to the best of our knowledge, not yet been used for end-capping of any CROP.In particular, PipMA proved to be well-suited for that purpose, as all DF were quantitative.For PipA, which represents a piperazinium salt with TFA, triethyl amine was added in situ to release the secondary amine as a nucleophile.DF values between 78 and 89% were reached.Variation of the excess of the termination agent or base did not increase the DF value further (data not shown).However, the nucleophilic attack of TFA on the living oxazolinium species was never observed.
In addition, the comonomer content was estimated from the 1 H NMR spectra based on signals assigned to the methyl protons of the EtOx repeating units.For cross-linkers containing MestOx, the overlapping signals of the POx backbone and the MestOx methyl group were used.In contrast, the methyl proton signals of the Boc moiety were well separated, simplifying the calculations for materials containing BocOx.In all cases, overlapping end group signals were taken into account as appropriate for the individual cross-linkers according to the assignments in Figures S16−S27.The resulting comonomer content was in good agreement with the feed ratio used in all cases (Table 3).The attachment of the various vinylic end groups was further confirmed by MALDI-TOF MS, as exemplified for crosslinkers derived from the homopolymerization of EtOx using the bifunctional initiator BIB in Figure 2. A repeating unit of 99 m/z was evident for all EtOx-based cross-linkers.The most abundant species were assigned to the desired structures, indicating a successful functionalization at all chain ends for all termination agents.Only the mass spectra of cross-linkers bearing PipA end groups featured a second pronounced m/z distribution, which is in accordance with the DF values described above.It was assigned to macromolecules comprising one PipA and one hydroxyl ω-end group.Analogous findings were made for the corresponding T-EtOx cross-linkers (see Figure S33 in the Supporting Information).
MALDI-TOF MS was also applied for the analysis of the cross-linkers containing MestOx or BocOx as comonomers (see Figures S34−S37 in the Supporting Information).For the MestOx-containing cross-linkers both repeating units for EtOx (Δm/z = 99) and MestOx (Δm/z = 157) were found confirming the successful synthesis of the copolymers.Accordingly, mass spectra of cross-linkers comprising BocOx revealed additional signals with a Δm/z = 228, which corresponds to the molar mass of one BocOx repeating unit.The elevated molar mass of MestOx and BocOx repeating units resulted in increased M n values for these cross-linkers compared to cross-linkers based on EtOx only (Table 3).For all cross-linkers, the end groups were confirmed by MS.
In summary, these characterization data show that welldefined POx can be obtained from bifunctional initiator BIB as well as trifunctional initiator TIB.Direct termination agents resulted in cross-linkers bearing radically polymerizable acrylate, methacrylate, acrylamide as well as methacrylamidebased end groups with high-end group fidelity.Values were determined using polystyrene (PS) as standard.b Measurements were carried out using trans-2-[3-(4-tert-butylphenyl)-2-methyl-2propenylidene] (DCTB) and sodium trifluoroacetate (NaTFA) as matrix mixture and poly(methyl methacrylate) (PMMA) for calibration.c Measurements were carried out using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix and poly(methyl methacrylate) (PMMA) for calibration.d Measurements were carried out using CDCl 3 as solvent.e Measurements were carried out using D 2 O as solvent.f Comonomer content was calculated from the corresponding monomer signals from 1 H NMR spectroscopy.

Deprotection of BocOx Containing Cross-Linkers.
A further functionalization of the cross-linkers via the amino moieties introduced by BocOx required the removal of the Boc protection groups.For this purpose, B-BocOx and T-BocOx oligomers were deprotected using trifluoroacetic acid (TFA) in order to obtain the corresponding B-Am and T-Am crosslinkers, respectively (Scheme 2). 1 H NMR spectroscopy confirmed the successful removal of the Boc protecting group for all cross-linkers (Figure 3).An overview about the signal assignments in the 1 H NMR spectra of the AmOx-based oligomers is given in the Supporting Information (see Figures S28−S32).
However, acrylate end groups were susceptible to cleavage from the cross-linkers during aqueous purification, both via extraction methods as well as by using anion exchange resins, as indicated by the loss of vinylic proton signals in the 1 H NMR spectra.Despite their ester functionality, methacrylate end groups were more stable so that TFA removal via a chloride-modified anion exchange resin was successful.In addition to 1 H NMR spectroscopy, MALDI-TOF MS clearly confirmed that these end groups were retained, although the DF was slightly lowered (Table 3 and Figures S38 and S39).precipitation in diethyl ether to result in a cross-linker TFA salt.The amount of TFA in B-Am-PipA and T-Am-PipA determined from 19 F NMR spectroscopy was in the expected range (5.0 or 8.5 TFA molecules per cross-linker molecule, respectively) (Figures S29 and S32).In contrast, the methacrylamide-based end group remained unaffected, even when aqueous purification methods were applied.However, the extraction using water and chloroform influenced the copolymer composition, as chains featuring a high amine content were susceptible to enrichment in the aqueous phase.B-Am-PipMA hence featured a lowered fraction of amino groups compared to that of the respective B-Boc-PipMA starting material. 19F NMR spectroscopy confirmed the removal of the excess of trifluoroacetic acid (Figures S28,  S30, and S31).
3.4.Cryogel Preparation and Characterization.The two-and three-armed EtOx-based cross-linkers with either acrylate, methacrylate, or PipA as the ω-end group were successfully used in the preparation of cryogels which were obtained as stable monoliths.Acryloyloxyethyl thiocarbamoyl rhodamine B (RhoB) was used as a functional comonomer to enable visualization of the gel structure by CLSM (Scheme 3).An overview about the reaction conditions used for the gel preparation based on both B-Et and T-Et cross-linker series is provided in Tables S2 and S3 in the Supporting Information.
Only for B-Et-PipMA and T-Et-PipMA, no or a reduced gel formation was observed, which resulted in a large amount of remaining liquid feed solution after the reaction.The poor polymerizability of N,N-dialkyl methacrylamides was already described by Suzuki et al. and might be due to steric effects of the alkyl substituents. 67Scanning electron microscopy (SEM) imaging revealed sponge-like morphologies throughout the entire series with median pore sizes from 24 up to 39 μm (Figures 4 and S40).Under the chosen conditions, pore sizes could be reproduced for all two-and three-armed cross-linkers with three different ω-end groups.According to the statistical analysis, significant differences between the pore sizes of most cryogels were found (Figure S41 and Table S4).However, no correlation between the pore sizes and either the end group or the number of polymer arms in the cross-linkers was found.
The visualization of the cryogel pore structures in the swollen state was also possible by the use of CLSM due to incorporated Rhodamine B. The confocal images revealed a homogeneous distribution of the fluorescent dye throughout the entire polymer network (Figure 5).A rough estimation of the pore sizes was possible from the magnified images.Based on these manual measurements for each of the six cryogels compared to SEM, an increase of the pore sizes in the native, hydrated state was found, with pore sizes ranging from 32 μm up to 92 μm.
Swelling experiments revealed significant differences in the water uptake ability among the cryogels (Figure 6 and Table S5).Cryogels based on trifunctional POx cross-linkers demonstrated a much slower swelling in comparison to their bifunctional analogues, except for the PipA containing polymers whose swelling ratios were almost identical.Presumably, this is due to the additional number of crosslinking points leading to an overall increasing cross-linking density/degree of cross-linking.Furthermore, acrylate and PipA containing cryogels reached the maximum swelling degree already after 20 s, whereas the methacrylate-containing analogues demonstrated a slower water uptake.This might be attributed to the additional methyl groups, which cause an increase in the hydrophobicity of the polymeric networks.However, all cryogels demonstrated a fast swelling behavior, which is comparable to PEGDA cryogels. 68n addition, no correlation between swelling and pore sizes for the individual cryogels was found. 13C ssNMR measurements revealed the presence of the different EtOx-based building blocks in the polymeric networks (see Figure S42 in the Supporting Information).No clear differences were visible within the spectra of cryogels based on either two-or three-armed macromonomers.In addition, all cryogels displayed excellent thermal stability up to at least 250 °C (see Figure S43 in the Supporting Information).Based on these results, the prepared cryogels are able to be sterilized by autoclaving (which usually proceeds at 120 °C) without material degradation prior to potential experiments with microorganisms or cells.When using B-Et-A and B-Et-MA, the first derivative TGA graphs displayed two major transition peaks at 355 and 415 °C or 304 and 417 °C, respectively.Notably, there was an intensity decrease of the earlier transition peak in the case of the B-Et-MA-based cryogels.In the case of B-Et-PipA-based cryogels, there is exclusively one transition peak at 428 °C, emphasizing the increased thermal stability of the piperazine-based ω-end group.
The use of three-armed instead of two-armed cross-linkers had no effect on the thermal stability of the cryogels, as shown by the almost identical curves.In addition, the stability of the prepared cryogels in aqueous solutions was investigated at different pH values (see Figure S44 in the Supporting Information).A complete dissolution of the cryogels containing acrylate ester bonds (B-Et-A, T-Et-A) was observed after 24 h under alkaline conditions.In contrast, methacrylate and PipA containing cryogel networks remained stable in a broad pH range even for 24 h.

CONCLUSIONS
We presented the development of cryogels that are solely based on cross-linkers derived from oligomeric 2-oxazolines.Thorough kinetic investigations of bifunctional and trifunctional benzyl iodides confirmed their excellent suitability as initiators for the CROP, confirming efficient and fast initiation at all sites.In addition to the successful incorporation of MestOx as well as BocOx as monomers comprising further functional moieties, the CROP could be terminated with four tailored nucleophiles, i.e., acrylate, methacrylate, piperazineacrylamide, and piperazine-methacrylamide. The resulting twoand three-armed POx-based cross-linkers featured high-end group fidelity.The attachment of the radically polymerizable moieties through ester as well as amide bonds generated cross- linkers and cryogels with tunable hydrolytic stability.Whereas cryogels cross-linked via piperazine-acrylamide moieties were stable in a broad pH value and temperature range, POxacrylate cross-linkers enabled access to materials that can be degraded under alkaline conditions.
Having developed access to the novel cryogels, our next steps include the exploitation of the broad range of materials that can now be made available.For instance, the varied crosslinking density will impact mechanical properties of the cryogels and could be further tuned by variation of the DP of the cross-linkers.In particular, cryogels consisting of MestOx and AmOx-based cross-linkers will be of interest for 3D cell culture as, e.g., peptides or sugars can be covalently attached to the materials.

■ ASSOCIATED CONTENT
* sı Supporting Information

Figure 1 .
Figure 1.Kinetic plots for the CROP of EtOx, EtOx and MestOx as well as EtOx and BocOx initiated by BIB ([M] 0 /[I] 0 = 10) and TIB ([M] 0 / [I] 0 = 15) carried out in acetonitrile ([M] 0 = 2 mol L −1 , reflux).Apparent polymerization rate coefficients (k p ) and polymerization rate coefficients per arm (k p,arm ) were calculated from the slope of the corresponding linear fits in the first-order kinetic plots and are summarized in Table1.Corresponding number-average molar masses (M n ) and dispersity values (Đ) as plotted against conversion were determined by SEC.

Figure 2 .
Figure 2. MALDI-TOF mass spectra (DCTB + NaTFA) of the different cross-linkers derived from the homopolymerization of EtOx using the bifunctional initiator BIB.From left to right: Full spectra, zoom into an m/z region displaying the EtOx repeating units, and overlay of the measured and calculated (red dotted line) isotopic pattern of the most abundant species (marked with *).All identified m/z species were found as sodium adducts.Scheme 2. Schematic Representation of the Deprotection of the BocOx-Containing Cross-Linkers Using BIB or TIB as Initiators (R 1 ) and Acrylate (A), Methacrylate (MA), PipA, or PipMA as the ω-End Group (R 2 )

Figure 3 .
Figure 3. Middle: Overlay of 1 H NMR spectra of the B-BocOx (black, CDCl 3 , 300 MHz) and the B-AmOx cross-linkers (red, D 2 O, 300 MHz).Left: Zoom into 1 H NMR spectra of the double bond signals.Right: Schematic representation of the chemical structures of the B-BocOx and B-AmOx cross-linkers.Scheme 3. Schematic Representation of the Synthesis of Cryogels by Cryo-Polymerization at −12 °C Based on Bi-and Trifunctional EtOx-Based Cross-linkers Bearing Various ω-End Groups (A, MA, PipA, and PipMA) a

Figure 4 .
Figure 4. SEM micrographs of cryogels based on B-EtOx and T-EtOx cross-linkers with different ω-end groups.For each type, an entire picture of the gel slice as well as a zoomed view (121×) is displayed.

Figure 6 .
Figure 6.Swelling ratios of the cryogel series based on different POx cross-linkers.Swelling experiments were performed in triplicate with the exception of CG-T-Et-MA (n = 2).

Table 1 .
Apparent Polymerization Rate Coefficients k p and Apparent Polymerization Coefficients per Initiation Site k p,arm Given in L mol −1 min −1 Calculated from the Slope of the Semi-Logarithmic Kinetic Plots According to Equations 2−4 polymer monomer k p [L mol −1 min −1 ] k p,arm [L mol −1 min −1 ]

Table 2 .
Overview on All Synthesized Cross-Linkers Based on BIB or TIB as Initiators, EtOx, and MestOx or BocOx as Monomers, Bearing Acrylate (A), Methacrylate (MA), and Piperazine-Based (PipA and PipMA) ω-End Groups Cross-linkers based on EtOx and AmOx were obtained after the deprotection of the BocOx containing analogues.b Loss of the ω-end group was observed by 1 H NMR spectroscopy. a

Table 3 .
Summary of All Relevant Analytical Data of Synthesized Cross-Linkers Obtained from SEC (M n , Đ), MALDI-TOF MS (M n and Đ), and a1 H NMR (Comonomer Content in %)