Synthesis and Solution Properties of Double Hydrophilic Poly(ethylene oxide)-block-poly(2-ethyl-2-oxazoline)

We demonstrate the synthesis of star-shaped poly(ethylene oxide)-block-poly(2-ethyl-2-oxazoline) [PEOm-b-PEtOxn]x block copolymers with eight arms using two different approaches, either the “arm-first” or the “core-first” strategy. Different lengths of the outer PEtOx blocks ranging from 16 to 75 repeating units were used, and the obtained materials [PEO28-b-PEtOxx]8 were characterized via size exclusion chromatography (SEC), nuclear magnetic resonance spectroscopy (NMR), and Fourier-transform infrared spectroscopy (FT-IR) measurements. First investigations regarding the solution behavior in water as a non-selective solvent revealed significant differences. Whereas materials synthesized via the “core-first” method seemed to be well soluble (unimers), aggregation occurred in the case of materials synthesized by the “arm-first” method using copper-catalyzed azide-alkyne click chemistry.


Introduction
The synthesis of polymer-based materials using different monomers, material compositions and macromolecular architectures can be realized via a multitude of synthetic methodologies.Mainly living and controlled polymerization techniques were developed to obtain polymers with narrow molar mass distributions, adjustable chain length and precisely positioned functional groups [1].Thereby, the architecture has a large influence on the physical properties of the final material.Moreover, the monomer distribution and composition along the polymer backbone directly influences the solubility and other physical properties [2][3][4].This has been demonstrated for random, gradient, graft and block copolymers synthesized by different polymerization techniques [5][6][7][8][9].In the case of linear homo-and (block) copolymers, the solution behavior has become quite predictable after a manifold of systematic studies for different monomer combinations and sequences during the last few decades [10][11][12][13][14]. On the other hand, the combination of polymer chains in one central point leads to star-shaped materials and can result in unprecedented morphologies, as well as solution behavior in selective and non-selective solvents [15][16][17][18].
Star-shaped amphiphilic block copolymers are of special interest in drug delivery applications, due to the absence of a critical micellar concentration (cmc, depending on the hydrophilic-to-hydrophobic balance of the system) and the possibility to take up and release suitable drugs.The "load" can be encapsulated into the inner part (core, hydrophobic) of the materials, while the outer shell (hydrophilic) stabilizes the system in, e.g., aqueous solution [19].If poly(ethylene oxide) (PEO) is used as the shell, "stealth"-behavior can be observed, also known as "PEGylation", preventing the recognition of such materials by our immune system.This renders such approaches suitable for the preparation of long-circulating polymer-based drug nanocontainers [20][21][22].
For the synthesis of star-shaped block copolymers, mainly two approaches have been employed, the divergent ("core-first") and the convergent ("arm-first") method [20,[23][24][25][26][27].The divergent approach uses a multifunctional initiator, but typically not all initiation sites are easily accessible, which drastically influences the number of arms and the overall degree of polymerization.Nevertheless, with increasing distance between the core and the initiation site, the initiation efficiency can be improved.Nevertheless, star-star coupling often occurs during, e.g., radical polymerizations, and limits the monomer conversion (arm length) in such attempts [2,4,28,29].As an alternative, the convergent approach employs pre-synthesized arms, which are subsequently connected to the core covalently in the final step, often providing superior control over arm length and number; moreover, an in-depth characterization of the constituting building blocks prior to joining the core and shell is possible.Such approaches have been described in the literature via supramolecular chemistry [24,30,31], metal-complexation [32] or click-chemistry [23,33,34].
Herein, we demonstrate the synthesis of star-shaped poly(ethylene oxide)-block-poly(2-ethyl-2oxazoline) [PEO m -b-PEtOx n ] x block copolymers with eight arms using two different approaches, either the "arm-first" or the "core-first" strategy.Regarding the core block, PEO-based materials of different architectures have been thoroughly investigated concerning solution behavior [35,36] or the possibility of being scaffolds in medical applications [19,20,22].The outer block, poly(2-ethyl-2-oxazoline) (PEtOx), is water-soluble and non-toxic, and the pseudo-peptide character of this material has been shown to induce similar "stealth" behavior, as observed for PEO [19,22,[37][38][39].PEtOx can be synthesized with a wide range of functional groups, being present via cationic ring-opening polymerization (CROP) [40][41][42][43].We used different lengths of the outer PEtOx blocks, and the obtained [PEO 28 -b-PEtOx x ] 8 materials were characterized via size exclusion chromatography (SEC), nuclear magnetic resonance spectroscopy (NMR) and Fourier-transform infrared spectroscopy (FT-IR).Whereas similar compositions could be prepared using either "core-first" or "arm-first" approaches, first investigations regarding the solution behavior in water as a non-selective solvent revealed significant differences.

Instruments
NMR: Proton nuclear magnetic resonance ( 1 H-NMR) spectra were recorded in CDCl 3 on a Bruker AC 300 MHz spectrometer at 298 K.Chemical shifts are given in parts per million (ppm, δ scale) relative to the residual signal of the deuterated solvent.Carbon NMR ( 13 C-NMR) spectra were recorded with 75 MHz.SEC: Size exclusion chromatography was measured on a Shimadzu system equipped with a SCL-10A system controller, an LC-10AD pump, an RID-10A refractive index detector and both a PSS Gram30 and a PSS Gram1000 column [Polymer Standards Services GmbH (Mainz, Germany)] in series, whereby N,N-dimethylacetamide (DMAC) with 5 mmol of lithium chloride (LiCl) was used as an eluent at a 1 mL min −1 flow rate.The column oven was set to 60 °C.The system was calibrated with polystyrene (PS; 100 to 1,000,000 g mol −1 ) standards.Furthermore, a Shimadzu system equipped with an SCL-10A system controller, an LC-10AD pump and an RID-10A refractive index detector using a solvent mixture containing chloroform (CHCl 3 ), triethylamine (TEA) and iso-propanol (i-PrOH) (94:4:2) at a flow rate of 1 mL min −1 on a PSS SDV linear M 5 μm column.The system was calibrated using PS (100 to 100,000 g mol −1 ) and PEO (440 to 44,700 g mol −1 ) standards.
MALDI-ToF MS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry was performed on an Ultraflex III TOF/TOF (Bruker Daltonics, Bremen, Germany), equipped with a Nd:YAG laser and with trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB) as the matrix and NaCl as the doping agent in reflector and linear mode.The instrument was calibrated prior to each measurement with an external poly(methyl methacrylate) (PMMA) standard from PSS Polymer Standards Services GmbH (Mainz, Germany).
FT-IR Infra-red spectroscopy: Dry powders of the copolymers were directly placed on the crystal of the ATR-FTIR (Affinity-1 FTIR, Shimadzu) for measurements in the range of 4000 to 600 cm −1 .
Microwave-assisted polymerizations were carried out utilizing an Initiator Sixty single-mode microwave synthesizer from Biotage, equipped with a non-invasive IR sensor (accuracy: 2%).Microwave vials (conical, 0.5 to 2 mL) were heated at 110 °C overnight and allowed to cool to room temperature under nitrogen atmosphere.All polymerizations were carried out using temperature control.DLS: Dynamic light scattering was performed at a scattering angle of 90° on an ALV CGS-3 instrument equipped with a He-Ne laser operating at a wavelength of 633 nm at 25 °C.Tetrahydrofuran (THF) [polytetrafluoroethylene (PTFE); 0.45 µm] and MilliQ-water [glass faser (GF); 1-2 µm] were filtered before usage.The CONTIN algorithm was applied to analyze the obtained correlation functions.For temperature control, the DLS is equipped with a Lauda thermostat.Apparent hydrodynamic radii were calculated according to the Stokes-Einstein equation.All CONTIN plots shown are number-weighted.SLS: For static light scattering (SLS), different concentrations between 1.5 and 3.5 mg mL −1 were prepared in THF and measured at 25 °C and different scattering angles (30° to 150°).Prior to the measurements, the samples and all solvents were filtered with PTFE-syringe filters (0.45 µm).
Liquid Chromatography under Critical Conditions (LCCC): High-performance liquid chromatography (HPLC) was measured on an Agilent system (series 1200) equipped with a binary pump, an autosampler and an evaporative light scattering detector (ELSD, Softa Corporation, Model 400).For the LCCC separation, a Nucleosil octadecylsilyl (ODS) column (Knauer, 100 mm × 3 mm, particle size 5 µm, pore size 100 Å) was used.The mobile phase consisted of a mixture of acetonitrile (ACN) and water (55/45, v/v) delivered by the binary pump at a flow rate of 0.5 mL min −1 .The column oven temperature was set to 45 °C.For the detection part, the ELSD was used with an evaporator temperature of 90 °C.The samples were dissolved at a concentration of 2 mg mL −1 in the same solvent mixture as the mobile phase and for each measurement, 20 μL were injected.The data was acquired using the WINGPC Unity software from PSS.To characterize the star-shaped PEO samples prior to 2D measurements, size exclusion chromatography (SEC) was measured separately on a Shimadzu system equipped with an SCL-10A system controller, an LC-10AD pump and an RID-10A refractive index detector using 100% THF as the solvent at a flow rate of 3 mL min −1 on a PSS-SDV-linear S column (PSS GmbH Mainz, 300 mm × 8 mm, particle size 5 μm) at 45 °C.The system was calibrated with PEO (M n = 1470 to 7000 g mol −1 ) standards purchased from PSS.
Two-dimensional liquid chromatography (2D-LC): For the first dimension LCCC of PEO, the analytical conditions were used as described above, except that the flow rate was set to 0.02 mL min −1 to enable the subsequent SEC separation of the LCCC fractions for the 2D analysis.The different sample fractions of the LCCC separation were automatically transferred to the second dimension (SEC) via an eight-port valve system with 100 μL sample loops.On the SEC system, the fractions were separated on an SDV HighSpeed linear S column from PSS (50 mm × 20 mm, particle size 5 μm) using THF as eluent at a flow rate of 3 mL min −1 at 45 °C and the ELSD.For the 2D measurements, higher concentrated polymer solutions (7 mg mL −1 ) were prepared, and 50 μL were used as the injection volume.The data acquisition was done by the PSS WINGPC unity software, including a 2D software module.
Transmission electron microscopy (TEM): The formed aggregates were analyzed using a TEM (Zeiss-CEM 902A, Oberkochen, Germany) operated at 80 kV.Images were recorded using a 1 k TVIPS FastScan CCD camera.TEM samples were prepared by applying a drop of an aqueous sample solution onto the surface of a plasma-treated carbon coated copper grid (Holey Carbon Grid + 2 nm C; Quantifoil Micro-Tools GmbH, Jena, Germany).

Tosylation of Star-Shaped [PEO 28 -OH] 8
The tosylation of [PEO 28 -OH] 8 (6 g; 0.6 mmol) was achieved in a slightly modified way as described in the literature [44,45].Briefly, the educts were dissolved in DCM and stirred at room temperature for at least 72 h, obtaining [PEO 28

Polymerization of 2-Ethyl-2-oxazoline using a Star-Shaped PEO Macroinitiator
For the polymerization of EtOx via a star-shaped macroinitiator [PEO 28 -Ts] 8 , different initiator to monomer ratios were chosen, and the polymerization was conducted in acetonitrile (1 M) in a microwave-synthesizer at 140 °C.The reaction was stopped via cooling the reaction mixture after 15 min and the addition of 0.2 mL of water.The final polymer was obtained via precipitation in THF at −30 °C.

Kinetic Investigation of the Polymerization of 2-Ethyl-2-oxazoline using a Star-Shaped PEO-Macroinitiator
A stock solution of the macroinitiator [PEO 28 -Ts] 8 and 2-ethyl-2-oxazoline (EtOx) were mixed with acetonitrile at a monomer to initiator ratio of 40 and a monomer concentration of 1 M.The capped vials were placed in a microwave synthesizer at 140 °C.The polymerization was terminated via the addition of water.The pure star-shaped block copolymers were received after precipitation in THF at −30 °C.

Results and Discussion
We were interested in the solution properties of well-defined star-shaped block copolymers containing two hydrophilic blocks.In particular, the influence of the length used for the outer block on the behavior in non-selective solvents (i.e., water) for a system with a given arm number (here: eight arms) was our focus for this study.We chose poly(ethylene oxide) as the core, due to its wide solubility in common solvents, its commercial availability and chemical inertness, enabling various chemical modifications.As the outer block (shell), we used poly(2-ethyl-2-oxazoline), a well-studied material with proven biocompatibility [22] and temperature-responsive properties (lower critical solution properties, LCST) above a threshold-molar mass in aqueous media [46,47].
Regarding the synthesis of star-shaped poly(ethylene oxide)-block-poly(2-ethyl-2-oxazoline) block copolymers with eight arms, we chose to compare two different strategies: for the "arm-first" approach, the macromolecular conjugation (azide-alkyne click reaction [27,48]) between azide-functionalized [PEO 28 -N 3 ] 8 and alkyne-functionalized TB-PEtOx x of different molar mass was used.In the case of the "core-first" strategy, tosylated [PEO 28 -Ts] 8 (the subscripts denote the degree of polymerization of the corresponding block, and the subscripts after the brackets represent the arm number of the herein described star-shaped block copolymers) was used as a macroinitiator for the cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazoline (EtOx).In both cases, the length of the PEtOx block can be easily varied within a certain range.In the following, first, both synthetic routes will be described separately, and afterwards, the solution properties in water as a non-selective solvent for both blocks will be compared.

Star Synthesis via Macromolecular Conjugation ("Arm-First"-Approach)
Core: First, a commercially available star-shaped poly(ethylene oxide) (PEO) with eight arms and a total molar mass (M n ) of 10,000 g mol −1 (1250 g mol −1 per arm) was modified.For this purpose, the hydroxyl end group was tosylated first by a nucleophilic substitution reaction using p-toluenesulfonyl chloride (Ts-Cl; Scheme 1), obtaining [PEO-Ts] 8 .Whereas this modification for linear PEO is often described as being performed within a few hours [44,45], in our case, the reaction time needed to be increased to at least 72 h at room temperature to achieve full functionalization (determined via 1 H-NMR; Figure S1C).Afterwards, [PEO 28 -Ts] 8 was converted to [PEO 28 -N 3 ] 8 using sodium azide (Scheme 1).After purification, for [PEO 28 -N 3 ] 8 , slight amounts (<5%) of residual aromatic signals, corresponding to the tosyl-moiety, were observed via 1 H NMR (Figure S1C).Nevertheless, the azide group could be clearly detected by ATR FT-IR measurements (2110 cm −1 ; Figure S1B).
To ensure full end-group conversion of the modified star-shaped macromolecules, the polymers were investigated via 13 C-NMR and 2D-LC (LCCC × SEC).In the latter case, liquid chromatography under critical conditions for PEO (LCCC) should enable the separation according to the end-group polarity and further coupled to SEC for the molar range [49][50][51][52][53].After careful adjustment of the critical conditions for PEO (Figure S2), the stars with different end-groups ([PEO 28 -OH] 8 , [PEO 28 -Ts] 8 and [PEO 28 -N 3 ] 8 ) were investigated (Figure 1).As can be seen, [PEO 28 -OH] 8 exhibits only one distribution, with a peak maximum at 0.62 mL (Figure 1A), whereas for [PEO 28 -Ts] 8 , two distributions at 0.80 mL (97%) and 0.62 mL (3%, unfunctionalized material) were observed (Figure 1B).This is in good agreement with the error of the NMR spectroscopy, where no [PEO 28 -OH] 8 could be detected.For [PEO 28 -N 3 ] 8 , also, two distributions at 0.80 mL (90%) and at 0.62 (10%) were observed (Figure 1C).We tentatively attribute this observation to a slow exchange of the azide functionality by hydroxyl groups under these conditions.This assumption can be confirmed by time-dependent investigations, leading to a ratio of 90:10 for both distributions directly after sample preparation, while 75:25 is found after 30 min (Figure S3).If the sample was stored overnight in solution, a ratio of 50:50 is observed.As many reactions using azides are described in the literature in water [49,54], this exchange process might be facilitated here, as the sample is heated to 45 °C and remains for 100 min within the system to elute/separate.The elution behavior in LCCC was similar for [PEO 28 -Ts] 8 and [PEO 28 -N 3 ] 8 , again, somewhat surprising.During SEC, further small distributions appear at higher elution volumes, which might be due to a higher flow rate of 3 mL min −1 (Figure 1D), as under flow rates of 1 mL min −1 , only monomodal distributions were observed.
Due to the results obtained for [PEO 28 -N 3 ] 8 using 2D-LC experiments, additional 13 C-NMR measurements in CDCl 3 were carried out.Here, the signals for the tosyl-group, as well as the CH 2 -group located next to the hydroxyl-end-group at 61.5 ppm are not observed, and also, two new signals for the two CH 2 groups next to the azide functionality at 50.5 and 79.8 ppm (Figure S4) confirm the successful conversion, at least within the experimental error of the NMR [45,55].
Arm: Alkyne-functionalized 2-ethyl-2-oxazoline homopolymers (TB-PEtOx x ) with different molar masses and low polydispersity indices (Ð; <1.1) were obtained via microwave-assisted cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazoline (EtOx) [25].Therefore, solutions containing a functional initiator, propargyl p-toluenesulfonate, with different monomer-to-initiator ratios ([M]/[I]) at a constant monomer concentration of 4 M were prepared and polymerized in a microwave-synthesizer at 140 °C.The degrees of polymerization (DP) obtained via 1 H NMR and MALDI-ToF MS slightly differ from the theoretically calculated values, according to the feed ratios used for the polymerizations.For a theoretical DP of 20, a DP of 18 (TB-PEtOx 18 ), for a DP of 60, a DP of 55 (TB-PEtOx 55 ), and for a DP of 80, a DP of 75 (TB-PEtOx 75 ) were found (Table 1, SEC in Figure S6A).The molar masses of the polymers increase linearly with the monomer-to-initiator ratio and are also in good agreement with the values obtained by MALDI-ToF MS measurements (Table 1).As the molar masses of the star-shaped block copolymers after arm attachment will exceed the exclusion volume of the utilized CHCl 3 SEC, the homopolymers (arms) were also subjected to another SEC instrument featuring a higher molar mass range (here, N,N-dimethylacetamide (DMAc) was used as the eluent, Figure S6B).The slight broadening of the Ð-values can be ascribed to polymer-column interactions, and, furthermore, the apparent molar masses are higher in comparison to the values obtained using chloroform as the eluent.
For the synthesis of [PEO 28 -b-PEtOx x ] 8 , star-shaped block copolymers [PEO 28 -N 3 ] 8 and different TB-PEtOx x were used in copper-catalyzed azide-alkyne cycloaddition reactions (CuAAC; Scheme 2).First, the conditions had to be optimized by variation of the solvent, the reaction temperature and the reaction time.The best conditions were obtained in a THF-ethanol mixture (1:1 v/v) at 80 °C using a four-fold excess of TB-PEtOx x in comparison to the azide-functionality and a reaction time of only 15 min in the microwave synthesizer.Under these conditions, it was possible to obtain the desired [PEO 28 -b-PEtOx x ] 8 materials.Purification was achieved via selective precipitation of the block copolymer in THF at −30 °C, while PEtOx x homopolymers are still soluble under these conditions.The obtained polymers were characterized using SEC (Table 2), FT-IR and NMR (Figure S7).Table 2. Selected characterization data for star-shaped [PEO 28 -b-PEtOx x ] 8 block copolymers obtained via the "core-first" or "arm-first" approach.
SEC elution traces using DMAC/LiCl (0.21 wt % LiCl) as the eluent for the individual building blocks and the star-shaped block copolymers are shown in Figure 2. The corresponding characterization data are shown in Table 2. Via this approach, three different star-shaped block copolymers, [PEO 28 -b-PEtOx 18 ] 8 , [PEO 28 -b-PEtOx 55 ] 8 and [PEO 28 -b-PEtOx 75 ] 8 , were obtained.Owing to the star architecture, the elution behavior of the star-shaped block copolymers leads to lower molar masses than expected during SEC measurements.This effect has already been described for other

Star Synthesis via CROP of 2-Ethyl-2-oxazoline Using a Star-Shaped Macroinitiator ("Core-First"-Approach)
Furthermore, here, commercially available [PEO 28 -OH] 8 was modified via tosylation as described above and purified until no further unreacted Ts-Cl could be observed in the 1 H-NMR spectra.As PEO is rather hydroscopic, the macroinitiator was co-evaporated with toluene, dried under vacuum for at least 24 h and stored in a glove box.After the preparation of the macroinitiator, we first carried out a kinetic study for the polymerization of EtOx (Figure 3).Therefore, a stock solution of [PEO 28 -Ts] 8 and monomer ([M]/[I] = 40) was prepared in ACN (1M) and divided into several microwave vials.The vials were subsequently placed in the microwave-synthesizer and analyzed after different polymerization times at 140 °C via 1 H-NMR and SEC.A pseudo-linear first-order kinetic was observed for the monomer consumption over time, while in SEC elution traces, two distributions were observed (Figure 3B).
Taking into account the slope of the fit in Figure 3A, a propagation rate (k p ) of 337 L mol −1 s −1 × 10 −3 can be calculated (corresponding to 42 L mol −1 s −1 × 10 −3 per arm).However, as indicated by the second distribution in the SEC elution traces in Figure 3B, with increasing polymerization time, also homopolymer (PEtOx) is formed, presumably due to transfer reactions.Although [PEO 28 -Ts] 8 has been extracted and co-evaporated with toluene several times prior to use, followed by drying for at least 24 h under vacuum, traces of impurities seem to persist.One way to determine the actual amount of incorporated PEtOx within the star-shaped [PEO 28 -b-PEtOx x ] 8 block copolymers is to remove the generated homopolymer via fractionated precipitation in THF at −30 °C.The results are depicted in Figure 4. Therefore, differences of up to 50% between the expected and the real PEtOx content can be observed.The monomer conversion obtained from the reaction solution seems to be up to 50% (DP = 20), but the monomer conversion determined via NMR from the purified product leads to 25% (DP = 10).Figure 4. Time-dependent EtOx conversion (black squares) and the corresponding degrees of polymerization per arm (red squares) determined from the reaction mixture (filled squares) and after purification of the star-shaped block copolymers (empty squares) via NMR (A); SEC traces before (dashed line) and after purification via fractionated precipitation (B) (solid lines; CHCl 3 was used as the eluent).
As can be seen in Figure 3B, after a polymerization time of 15 min, a considerable and clear shift of the desired product is visible in the elution traces and, at the same time, the amount of homopolymer formed is mediocre.The overall monomer conversion is around 50%, and we chose this as the conditions for the synthesis of samples with different PEtOx block lengths.Due to the fact that not many differences were observed between [PEO 28 -b-PEtOx 55 ] 8 and [PEO 28 -b-PEtOx 75 ] 8 (Table 2), according to SEC measurements, [PEO 28 -b-PEtOx 20 ] 8 and [PEO 28 -b-PEtOx 60 ] 8 were targeted using the "core-first" approach, and the corresponding polymerizations were stopped at around 50% monomer conversion.The results are summarized in Table 2.In the case of the purified [PEO 28 -b-PEtOx 50 ] 8 , static light scattering (SLS) in THF was used in addition for the determination of the absolute molar mass (M W ). While in theory, a molar mass of 50,000 g mol −1 would be expected for [PEO 28 -b-PEtOx 50 ] 8 by the [M]/[I]-ratio and NMR, SLS leads to a value of 54,000 g mol −1 , being in quite good agreement (Table 2).
We also compared the elution volume of star-shaped block copolymers with similar composition, but synthesized via two different approaches (Figure 5).As can be seen, for systems with a similar DP of roughly 20, the elution behavior is comparable via SEC (Figure 5A), as in NMR, the DP for the "arm-first" approach was 16, compared to 18 in the case of the "core-first" sample.For the star block copolymer with a higher amount of PEtOx (DP of 50) , a shift to lower elution volume for the "core-first" product can be seen.Here, actual degrees of polymerization of 55 ("arm-first") and 50 ("core-first") for PEtOx were determined.

Study of Star-Shaped [PEO 28 -b-PEtOx x ] 8 in Non-Selective Solvents
We were now interested in the solution properties of star-shaped [PEO 28 -b-PEtOx x ] 8 block copolymers in non-selective solvents, for example, tetrahydrofuran (THF) or water.First, the hydrodynamic radii in solution were determined using dynamic light scattering (DLS).Therefore, the samples were dissolved in THF, filtered (0.45 µm, PTFE) and the size was compared to the crude [PEO 28 -OH] 8 star polymer (Figure 6).According to the CONTIN plots depicted in Figure 6A, for [PEO 28 -OH] 8 , an apparent hydrodynamic radius of <R h > n,app = 1.5 nm was observed, whereas for the star-shaped block copolymers prepared via the "arm-first" approach, apparent hydrodynamic radii of 2.5 ([PEO 28 -b-PEtOx 18 ] 8 ), 4 nm ([PEO 28 -b-PEtOx 55 ] 8 ) and 5 nm ([PEO 28 -b-PEtOx 75 ] 8 ) were determined under these conditions (Table 3).These results, in our opinion, both confirm the formation of unimers in THF and the elution behavior observed in SEC with increasing length of the outer PEtOx block.The hydrodynamic radii obtained for "core-first" [PEO 28     However, if these "arm-first" materials are directly dissolved in water, again, a non-selective solvent for both PEO and PEtOx, turbid solutions are obtained.Transferring the materials from THF to water, via dialysis or evaporation of the organic co-solvent, leads to the same result.The turbidity did not decrease after heating (up to 100 °C), cooling (~5 °C for one week), changing the pH (0 to 12), prolonged sonication or the addition of different salts (e.g., KSCN, NaCl, KCl).For these turbid solutions, hydrodynamic radii of up to several hundred nm were observed, even at very low concentrations (<0.5 g L −1 , Table 3, Figure S8).At this point, we assume that this turbidity originates from the aggregation of the star-shaped block copolymers, although both blocks are of hydrophilic nature.Such behavior has also been described for water-soluble homo-and block copolymers in the literature [12,19,[56][57][58].In some cases, the unexpected aggregation of double-hydrophilic block copolymers was explained by slight differences in the hydrophilicity of both blocks [50,58].
If, on the other hand, star-shaped [PEO 28 -b-PEtOx x ] 8 block copolymers synthesized via the "core-first" approach were treated the same way, clear aqueous solutions with hydrodynamic radii of ~3 nm (both cases) are obtained.Somehow, the effect of aggregation in aqueous media is limited to samples prepared by click chemistry, for which we have no conclusive explanation up to now.No detectable amounts of copper were found in atom absorption spectroscopy (AAS), and therefore, an influence of residual copper from the CuAAC reaction can be excluded.
Applying shear forces via filtration (syringe filter, 1 µm, GF) to the turbid solutions of all described "arm-first" samples leads to clear solutions.To ensure that no material was removed by filtration, a defined concentration was filtered and dried afterwards, and the weight loss was below 5%.In Figure 6B, the DLS CONTIN plots for [PEO 28 -OH] 8 (dashed black line, <R h > n,app = 3 nm), [PEO 28 -b-PEtOx 18 ] 8 (red line, <R h > n,app = 6 nm) and [PEO 28 -b-PEtOx 75 ] 8 (blue line, <R h > n,app = 14 nm) are depicted.The obtained size distributions by DLS are slightly larger if compared to THF (<R h > n,app = 1.5 nm, 2.5 nm and 5 nm), respectively.This might be an indication for the formation of aggregates by entanglements or that the star-shaped block copolymers are highly swollen.
It is well known that PEtOx materials exhibit lower critical solution temperatures (LCST), depending on the chain length [47,59].To probe this for the herein described star-shaped systems, solutions of [PEO 28 -b-PEtOx 20 ] 8 and [PEO 28 -b-PEtOx 80 ] 8 (2.5 mg mL −1 , non-filtered aqueous solution) were heated up to 100 °C, and the turbidity was recorded.In both cases, the solutions did not show cloud points.We ascribe the absence of LCST behavior to the presence of a double-hydrophilic system and, in the case of [PEO 28 -b-PEtOx 20 ] 8, to the short PEtOx arms.
As another peculiarity, it has been reported by Demirel et al. that PEtOx with a molar mass of 500 kg mol −1 (1 mg mL −1 ) crystallizes after being heated in dilute solutions for several days at 70 °C [60].We therefore were interested in whether similar observations can be made for star-shaped systems of different composition.Solutions of [PEO 28 -b-PEtOx 18 ] 8 and [PEO 28 -b-PEtOx 75 ] 8 were heated to 80 °C in water for three days.No changes could be detected for the materials synthesized using the "core-first" approach, whereas larger aggregates were found by DLS and transmission electron microscopy (TEM) for [PEO 28 -b-PEtOx 18 ] 8 and [PEO 28 -b-PEtOx 80 ] 8 prepared via the "arm-first" methodology (Figure 7, only if unfiltered solutions were used).The structure of such aggregates was different, depending on whether [PEO 28 -b-PEtOx 18 ] 8 (55 wt % PEtOx) or [PEO 28 -b-PEtOx 75 ] 8 (82 wt % PEtOx) was used.In the case of [PEO 28 -b-PEtOx 18 ] 8 , sharp, crystal-like structures were observed, possibly due to partial crystallization of PEtOx, which was also observed by Güner et al., [60] leading to an alignment in a rod-like fashion (Figure 7A).Assemblies of several hundred nm in length and ~200 nm width were obtained.For [PEO 28 -b-PEtOx 75 ] 8 , a slightly different aggregation mechanism might take place: here, the superstructures, rather, look like micellar clusters, which might be the result of an initial formation of unimolecular micelles, followed by further agglomeration (Figure 7B) [61,62].Tentatively, the hydrophilicity of PEtOx might be lower in comparison to PEO; we assume a partial collapse of the outer PEtOx shell over time, leading to aggregation.However, as this effect was only observed for star-shaped block copolymers prepared via the "arm first" approach and only if non-filtered solutions were used, we hypothesize that a certain pre-organization is required.The aggregation behavior of PEO depending on the treatment before was also discussed by Güner et al. for linear polymers [56], and differences in hydrophilicity were described by Ke et al. [50,60].

Conclusions
We demonstrated the synthesis of a series of well-defined star-shaped [PEO 28 -b-PEtOx x ] 8 block copolymers using two different approaches, either "core-first" using [PEO 28 -Ts] 8 as a macroinitiator for the CROP of EtOx or by copper-catalyzed azide-alkyne cycloadditions between [PEO 28 -N 3 ] 8 and TB-PEtOx x ("arm-first").In both cases, different block lengths for the outer PEtOx block were used, and comparable molar masses and hydrodynamic radii in THF as a non-selective solvent were observed.In dilute aqueous solutions, samples prepared via the "arm-first" approach showed aggregate formation, whereas this was not the case for the "core-first" materials.Although this behavior is not fully understood at this point, we could exclude several factors (salt, copper impurities, differences in the molar mass) as the origin of this peculiarity, and we could show that after filtration (1 µm pore size), also, here, smaller hydrodynamic radii are found.At this point, our hypothesis is that small differences in hydrophilicity are the main driving force for this behavior.More importantly, such loose aggregates from star-shaped block copolymers could be used for the temperature-induced formation of larger agglomerates, where first investigations hint towards an influence of the weight ratio PEO:PEtOx on the morphology of the superstructures formed.

Figure 3 .
Figure 3. First order time-conversion plot for the kinetic investigation of the microwave assisted polymerization of EtOx with [PEO 28 -Ts] 8 as the initiator at 140 °C (A); comparison of the time-dependent SEC traces (CHCl 3 ) for the polymerization of EtOx (B).
CONTIN plot; b CONTIN plots in the Supporting Information part Figure S7; c non filtered sample.

Table 3 .
Determination of the apparent hydrodynamic radius (<R h > n,app ) for different star (block co-) polymer systems in non-selective solvents via DLS.