Core-crosslinked worm-like micelles from polyether-based diblock terpolymers

Johanna K. Elter ab, Philip Biehl ab, Michael Gottschaldt ab and Felix H. Schacher *ab
aInstitute of Organic Chemistry and Macromolecular Chemistry, Friedrich Schiller University Jena, Humboldtstraße 10, D-07743 Jena, Germany. E-mail: felix.schacher@uni-jena.de
bJena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, D-07743 Jena, Germany

Received 16th July 2019 , Accepted 13th September 2019

First published on 16th September 2019


We herein report on the synthesis, purification and subsequent self-assembly of different polyether-based diblock terpolymers in aqueous solutions. While in all cases PEO was used as hydrophilic macroinitiator in anionic ring opening polymerization (AROP), the hydrophobic block was formed by different hydrophobic glycidyl ethers, thereby and by variation of the DP also influencing micellar morphologies in water as selective solvent. The application of sterically demanding, branched aliphatic glycidyl ethers (2-ethylhexyl glycidyl ether (EHGE), or hexyl, hexyl glyceryl glycidyl ether (HHGGE)) or aromatic glycidyl ethers (benzyl glycidyl ether (BGE) or naphthyl glycidyl ether (NGE)) as main comonomer for the hydrophobic block showed distinct influence on the morphologies formed by self-assembly in solution. In general, we observed a comparably broad window in the corresponding phase diagram for the formation of worm-like structures. Further, 10% of furfuryl glycidyl ether (FGE) was incorporated in the hydrophobic block of the diblock terpolymers to enable subsequent crosslinking of the micellar core. Hereby, thermally induced Diels–Alder reactions using a hydrophobic bismaleimide which was encapsulated in the micellar core prior to crosslinking could be successfully employed. We also carried out first experiments regarding ultrasound treatment of the crosslinked aggregates, revealing the formation of branched worm-like structures from previously well-defined filomicelles. All solution structures as well as the experiments on crosslinked worm-like micelles were investigated via light scattering (DLS) and transmission electron microscopy (cryo-TEM).


Introduction

Amphiphilic block copolymers, i.e. block copolymers containing both hydrophobic and hydrophilic segments, are capable of forming aggregates consisting of a hydrophobic core and a hydrophilic corona in aqueous media similar to small-molecule surfactants.1 In contrast to the latter, well-defined macromolecules can form distinct nanostructures whose size and shape is controllable by well-considered choice of molecular weight and block length ratios of the underlying block copolymer,2,3 monomer structure,4 and preparation technique of the micelles.5,6 Due to their variability, such block copolymer micelles are of great interest in the context of medicinal chemistry, as they can be used as nanocarriers in drug or gene delivery approaches.7–10

In polymer therapeutics, biocompatibility of the materials is of great importance. Therefore, poly(ethylene oxide) (PEO) is often used as hydrophilic building block,9 as it exhibits low toxicity as well as low recognition by the immune system.11 As a hydrophobic block, poly(glycidyl ethers) bear the possibility to introduce a variety of side groups connected to a flexible polyether backbone.12 Thus, not only control over the micellar morphology can be exerted, the choice of specific hydrophobic comonomers also allows to introduce functional groups for subsequent functionalization,13,14 encapsulation of specific guest molecules, or crosslinking of the micellar core15,16 by post-polymerization modification reactions. Core crosslinking of micelles can be of special interest in biomedicine to “freeze” specific micellar shapes which show favorable cell uptake or clearance behavior.17,18 The shape can be preserved even if the micellar environment is changed or the micelle is exposed to strong shear forces in the blood stream. Crosslinking can be carried out for example via Diels–Alder reactions,15 thiol–ene and azide–alkyne click reactions,19 or UV-crosslinking of allyl groups20 incorporated within the micellar core. Finally, stimuli-responsive groups can be introduced to enable directed disassembly or secondary aggregation of the micelles formed from the respective block copolymers.10,21,22

As narrow molar mass distributions and pure products are often considered as crucial for the formation of uniform micelles from block copolymers, anionic polymerization is typically the method of choice in this field.23 In that regard, the polymerization of ethylene oxide (EO) as well as glycidyl ethers (GEs) via anionic ring opening polymerization (AROP) is well established.24 The synthesis of amphiphilic block copolymers can be carried out as a one-pot reaction by sequential addition of the desired monomers25 or as a multi-step synthesis including potential purification steps after block extension.18 In both cases, low dispersities and well-defined compositions can be achieved. If necessary, side products caused by transfer reactions occurring at high molecular weights of growing polymer chains can be removed using suitable purification procedures.26,27

In this work, we present the synthesis of AB diblock terpolymers containing a PEO segment as hydrophilic block and a hydrophobic block formed from four different hydrophobic glycidyl ether monomers and 10% of furfuryl glycidyl ether (FGE) as crosslinkable comonomer. All diblock terpolymers obtained were characterized via size exclusion chromatography (SEC) and 1H-NMR. The different comonomers used as well as the degree of polymerization (DP) of the hydrophobic block showed a distinct influence on the morphology of the nanostructures obtained upon micellization in water as selective solvent, especially with regard to the formation of worm-like micelles. Such nanostructures are of special interest for biomedical applications, as they can show extended circulation time in the blood stream and a higher drug loading capacity.28–32 Also, enhanced uptake in tumor cells as well as tissue penetration was shown in various in vitro and in vivo studies.33,34 Besides that, filomicelles also find application in hydrogels as future cell culture medium as well as in immunotherapy.35,36 Therefore, we focused on the generation of diblock terpolymers showing a comparably large window for worm-like structures in the corresponding phase diagram. In that regard, we also investigated various hydrophobic (co)monomers and their influence on structure formation, besides introducing a reactive allyl end group for subsequent surface functionalization of the generated micelles.

While block copolymers incorporating 2-ethylhexyl glycidyl ether formed filomicelles of several micrometers in length at weight fractions of the hydrophobic block ranging from 46% to 56% and shorter worm-like structures and spheres at a weight fraction of 31%, hexyl, hexyl glycidyl ether led to different micellar morphologies from spherical micelles at a weight fraction of 29% over pure filomicelles at 46% to mixed fractions of filomicelles and large vesicles at higher hydrophobic block weight fractions. Micelles were prepared by direct dissolution of the diblock terpolymers in water and were characterized via dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). Crosslinking of the micellar structures was carried out by incorporation of a bismaleimide crosslinker within the micellar core and subsequent Diels–Alder reactions with the furfuryl units of the hydrophobic block. Successful crosslinking was proven by DLS measurements. Further, first experiments on the influence of ultrasonication on length and morphology of the crosslinked, worm-like aggregates with flexible cores were carried out. It is known in literature that the length of filomicelles containing a crystalline core can be tuned by the application of ultrasound and that short filomicelles as well as micellar seeds could be produced by application of different sonication times and methods.37,38 Additionally, external stimuli like temperature can also influence the contour length of filomicelles.39 In contrast to that, size control for filomicelles with an amorphous core is less frequently investigated, one example being the influence of shear forces during the preparation process.5

Experimental part

Materials

All starting materials were purchased in analytical grade from Sigma-Aldrich (BGE, FGE, NaH, mCPBA, isopropylidene glycerine, TBABr, HBr, paraformaldehyde), VWR Chemicals (THF, MeOH, EtOH, AcOH, isopropanol, hexane, diethyl ether), Fisher Scientific (1-bromohexane, DCM, allyl chloride), Carl Roth (KOH, DMF), Grüssing (H3PO4), j&k (EHGE), Acros Organics (KH), or Alfa Aesar (allyl alcohol, bismaleimide) and were used as received if not mentioned otherwise. Solvents used for anionic polymerization were stirred with sodium and benzophenone at room temperature until the blue color of the benzophenone ketyl radical indicated the absence of traces of water. Subsequently, the solvents were distilled from sodium in a still apparatus and stored at room temperature over 3 Å molecular sieves under argon in a glovebox. EO was purchased in small steel bottles from Linde and distilled prior to use. All glycidyl ethers were stirred over CaH2 for at least 24 hours, subsequently distilled and stored at −21 °C under argon in a glovebox if not mentioned otherwise. TBABr was recrystallized from EtOAc and stored dry. Any glassware was cleaned in a KOH/isopropanol bath and dried at 100 °C. All deuterated solvents were obtained from Eurotops and ABCR. The micropure water having a conductivity of 0.86 μS cm−1 was taken from a TKA MicroPure UV System (JWT GmbH) connected to a desalination system (HLA 2800 M-3, Leyco Wassertechnik GmbH).

Methods

An Agilent 1260 Infinity system equipped with a 1260 IsoPump (G1310B), a 1260 ALS (G1310B) autosampler and three consecutive PSS SDV, 5 μm, 8 × 300 mm columns was used for SEC measurements. THF was used as an eluent at a flow rate of 1 mL min−1. The column oven was set to 30 °C and signals were detected using a 1260 DAD VL (G1329B) and a 1260 RID (G1315D) detector. The system was calibrated using PSS PEG/PEO (238 to 969[thin space (1/6-em)]000 g mol−1) standards.

1H-NMR measurements were performed on a 300 MHz Bruker spectrometer using CDCl3 or acetone-d6 as deuterated solvent. For calibration the specific signals of the non-deuterated species were used. HR-MAS NMR spectra were collected on a 500 MHz Bruker Avance III HD spectrometer equipped with a 4 mm dual 1H/13C HR-MAS probe head. Spinning frequency was set to 5 kHz. CDCl3 was used as a deuterated solvent for swelling of the samples. The residual solvent peak was used for referencing.

Dynamic light scattering (DLS) measurements were performed on an ALV DLS/SLS equipment consisting of an ALV Laser CGS3 goniometer with an ALV Avalanche correlator and a He–Ne laser (λ = 632.8 nm). For aqueous samples, solvent viscosity and refractive index were automatically adjusted to the temperature of the thermostat. For samples in THF/water mixtures, solvent viscosity and refractive index were calculated from literature values for 298 K. The CONTIN algorithm was applied to analyze the obtained correlation functions. Apparent hydrodynamic radii were calculated according to the Stokes–Einstein equation.

Cryo-transmission electron microscopy (TEM) measurements were performed on a FEI Tecnai G2 20 cryo-transmission electron microscope. Acceleration voltage was set to 200 kV. Samples were prepared on Quantifoil grids (3.5/1) after cleaning by argon plasma treatment for 120 s. 8.5 μL of the solutions were blotted by using a Vitrobot Mark IV. The aqueous samples (1 mg mL−1) were plunge-frozen in liquid ethane and stored under nitrogen before being transferred to the microscope utilizing a Gatan transfer stage. TEM images were acquired with a 200 kV FEI Tecnai G2 20 equipped with a 4k × 4k Eagle HS CCD and a 1k × 1k Olympus MegaView camera. Micrographs were adapted in terms of brightness and contrast using the software ImageJ 1.47v.

Small angle X-ray scattering (SAXS) was performed on a Bruker AXS Nanostar U (Bruker, Karlsruhe, Germany) equipped with a microfocus X-ray source (Incoatec IμSCu E025, Incoatec, Geesthacht, Germany), and operating at λ = 1.54 Å. The sample-to-detector distance was 107 cm and a pinhole setup with 750 μm, 400 μm, and 1000 μm was used. The thin film was mounted on a metal rack and fixed using tape. The domain size was calculated using Bragg's equation:

image file: c9py01054h-t1.tif

Ultrasonication was perfomed using a Sonic VibraCell VC505 Ultrasonic Processor (Sonics & Materials Inc.). The net output of the device was 500 Watt with a frequency of 20 kHz and tunable amplitude. The sonication tip was enclosed in a sound abating housing.

Synthesis of hexyl, hexyl glyceryl glycidyl ether (HHGGE, 2)

DL-1,2-Isopropylideneglycerol (7,5 mL, 60.53 mmol), tetra-n-butylammonium bromide (122 mg, 0.378 mmol) and KOH (5.1 g, 90.80 mmol) were dissolved in 80 mL of n-hexane in a Schlenk flask under argon. Subsequently, allyl chloride (9.9 mL, 121.06 mmol) was added via syringe through a septum. Then, the suspension was stirred under argon atmosphere at 40 °C for 20 hours. Afterwards, the mixture was washed with water (2 × 100 mL) and the organic phase was dried over Na2SO4. The solvent was evaporated under reduced pressure and the crude product was purified via column chromatography (n-hexane/diethyl ether 4[thin space (1/6-em)]:[thin space (1/6-em)]1) to yield 5.4 g (52%) of 4-((allyloxy)methyl)-2,2-dimethyl-1,3-dioxolane as colorless oil.

1H NMR (300 MHz, CDCl3) δ = 5.87 (ddt, J = 17.2, 10.4, 5.7 Hz, 1H), 5.24 (ddd, J = 17.2, 3.2, 1.6 Hz, 1H), 5.15 (ddd, J = 10.4, 2.8, 1.2 Hz, 1H), 4.35–4.16 (quint, J = 6.0 Hz, 1H), 4.08–3.97 (m, 3H), 3.70 (dd, J = 8.2, 6.4 Hz, 1H), 3.45 (ddd, J = 23.9, 9.8, 5.6 Hz, 2H), 1.39 (s, 3H), 1.33 (s, 3H) ppm.

In the next step, 4-((allyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (5.4 g, 31.36 mmol) was dissolved in 75 mL of a 4[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture of acetic acid and water. The mixture was stirred at 50 °C for 6 hours. Then, the solvent was removed under reduced pressure. The crude product 3-allyloxy-1,2-propanediol (4.0 g, 97% yield calculated by weight) was directly used in the next step.

1H NMR (300 MHz, CDCl3) δ = 5.92 (ddt, J = 16.1, 10.9, 5.7 Hz, 1H), 5.29 (ddd, J = 6.9, 3.5, 2.0 Hz, 1H), 5.22 (ddd, J = 10.4, 3.0, 1.3 Hz, 1H), 4.04 (dt, J = 5.7, 1.1 Hz, 2H), 3.96–3.87 (m, 1H), 3.77–3.68 (m, 1H), 3.67–3.56 (m, 1H), 3.58–3.45 (m, 2H) ppm.

In the third step, 3-allyloxy-1,2-propanediol (3.3 g, 25.27 mmol) was dissolved in 170 mL of DMF under argon atmosphere. Then, NaH (60% dispersion in mineral oil, 6.7 g, 167.32 mmol) was added portion wise to the solution. Subsequently, 1-bromohexane (8.8 mL, 63.26 mmol) was added via syringe. The suspension was stirred at room temperature for 72 hours. After that, the reaction mixture was quenched with 400 mL of methanol. The solvents were removed under reduced pressure and the residue was dissolved in 100 mL of ethyl acetate. The solution was washed with water (2 × 200 mL) and brine (1 × 100 mL) and then dried over Na2SO4. The solvent was removed under reduced pressure to yield 7.6 g (100% yield calculated by weight) of the crude product 3-allyloxy-1,2-hexyloxy propane as colorless oil, which was used in the next step without further purification.

1H NMR (300 MHz, CDCl3) δ = 6.01–5.83 (ddt, J = 17.4, 10.5, 5.7 Hz, 1H), 5.28 (ddd, J = 17.3, 3.3, 1.6 Hz, 1H), 5.18 (ddd, J = 10.4, 3.5, 1.5 Hz, 1H), 4.03 (dt, J = 5.5, 1.3 Hz, 2H), 3.69–3.38 (m, 9H), 1.57 (m, 4H), 1.44–1.15 (m, 12H), 1.03–0.77 (m, 6H) ppm.

In the last step, 3-allyloxy-1,2-hexyloxy propane (7.3 g, 24.44 mmol) was dissolved in 180 mL of DCM and cooled to 0 °C. Then, a solution of m-chloroperoxybenzoic acid (11.6 g, 87.96 mmol) in 180 mL of DCM was added dropwise over 20 minutes. The reaction mixture was then stirred for 48 hours while slowly warming to room temperature. Subsequently, the reaction solution was washed with an aqueous 1 N NaOH solution (1 × 200 mL) and water (2 × 200 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified via column chromatography (ethyl acetate/n-hexane 1[thin space (1/6-em)]:[thin space (1/6-em)]2) to yield 4.6 g (60%) of the desired product 2,3-hexyloxy-1-(oxiranylmethoxy) propane (hexyl, hexyl glyceryl glycidyl ether, HHGGE, 2) as a colorless oil. The oil was further dried by stirring at room temperature under vacuum for three hours before it was stored at −21 °C under argon in a glove box.

1H NMR (300 MHz, CDCl3) δ = 3.78 (ddd, J = 11.6, 3.1, 1.5 Hz, 1H), 3.70–3.37 (m, 11H), 3.23–3.11 (m, 1H), 2.81 (dd, J = 5.0, 4.2 Hz, 1H), 2.62 (dt, J = 5.3, 2.8 Hz, 1H), 1.65–1.49 (m, 4H), 1.46–1.21 (m, 12H), 0.90 (t, J = 6.7 Hz, 6H) ppm.

13C NMR (75 MHz, CDCl3) δ = 77.88, 72.08, 71.67, 71.60, 70.63, 70.53, 50.82, 50.79, 44.28, 44.25, 31.67, 30.03, 29.61, 25.78, 25.74, 22.61, 14.02 ppm.

Synthesis of 1-methylnaphthyl glycidyl ether (NGE, 3)

In a first step, naphthalene (12.8 g, 0.1 mol) and paraformaldehyde (6.0 g, 0.2 mol) were dissolved in a mixture of glacial acetic acid (12.4 mL, 0.22 mol) and phosphoric acid (85%, 8 mL, 0.12 mol). Then, concentrated hydrobromic acid (48%, 30 mL, 0.26 mol) was added dropwise to the suspension at room temperature. Afterwards, the reaction mixture was stirred at 80–85 °C for 6 hours. The resulting yellow suspension was left stirring overnight at room temperature. Then, the reaction mixture was extracted with chloroform three times. The combined organic layers were washed with water and 10% aqueous potassium carbonate, dried over sodium sulfate and filtered off. Evaporation of the organic solvent yielded a yellow oil (22 g, 99.5% yield calculated by weight). The crude product was directly used in the next step.

1H NMR (300 MHz, acetone) δ = 8.24 (d, J = 8.6 Hz, 1H), 7.95 (dd, J = 12.8, 8.0 Hz, 2H), 7.70–7.41 (m, 5H), 5.14 (s, 2H).

In the next step, allyl alcohol (5 mL, 73.17 mmol) was dissolved in 50 mL of dry THF under argon in a three-necked flask equipped with a reflux condenser and a thermometer. Sodium hydride (95%, 2.4 g, 95.13 mmol) was suspended in 30 mL of dry THF and added slowly to the solution. Then, the mixture was heated to 70 °C for 30 minutes. Afterwards, the reaction mixture was cooled to 0 °C in an ice-bath. Crude 1-bromomethyl naphthalene (19 g) dissolved in 80 mL of dry THF was added dropwise via septum. Then, the reaction was allowed to slowly warm to room temperature. Stirring was continued for 6 h before 50 mL of methanol were added to quench residual sodium hydride. The solvents were removed under reduced pressure and the residual red solid was dissolved stepwise in DCM. The organic phases were washed with brine (3×) and dried over Na2SO4. The salt as well as remaining solids were then filtered off and the solvent was removed under reduced pressure to yield 15.8 g of the crude product as bright orange oil. The crude product was purified via column chromatography (hexane/diethyl ether 20[thin space (1/6-em)]:[thin space (1/6-em)]1 → 8[thin space (1/6-em)]:[thin space (1/6-em)]1) yielding 4.3 g (30%) of 1-((allyloxy)methyl)naphthalene as yellow oil. Additionally, 5.3 g (33%) of the reactant 1-bromomethyl naphthalene were recovered as light yellow oil.

1H NMR (300 MHz, acetone-d6) δ = 8.21–8.14 (m, 1H), 7.97–7.84 (m, 2H), 7.61–7.44 (m, 4H), 6.01 (ddt, J = 17.2, 10.6, 5.4 Hz, 1H), 5.34 (ddd, J = 17.3, 3.6, 1.7 Hz, 1H), 5.19 (ddd, J = 10.4, 3.3, 1.4 Hz, 1H), 4.98 (s, 2H), 4.12 (dt, J = 5.4, 1.5 Hz, 2H) ppm.

In the next step, 1-((allyloxy)methyl)naphthalene (4.3 g, 21.64 mmol) was dissolved in 125 mL of dry DCM and cooled to 0 °C. Then mCPBA (ca. 77%, 15.0 g, 66.93 mmol) was dissolved in 75 mL of dry DCM and added dropwise to the solution under stirring. The mixture was allowed to warm to room temperature afterwards and stirring was continued for three days. Subsequently, the reaction solution was diluted to 50 mL with DCM and washed with 1N NaOH (100 mL), water (100 mL) and brine (50 mL). The aqueous phase was extracted again with 25 mL of DCM. The organic phases were combined, dried over MgSO4 and the solvent was removed under reduced pressure, yielding 4.0 g of the crude product as yellow oil. The crude product was purified via column chromatography (ethyl acetate/hexane 1[thin space (1/6-em)]:[thin space (1/6-em)]5 → 1[thin space (1/6-em)]:[thin space (1/6-em)]3), yielding 1.7 g (37%) of the desired product 2-((naphthalen-1-ylmethoxy)methyl)oxirane (1-methylnaphthyl glycidyl ether, NGE, 3). The low yield after column chromatography might be a result of decomposition of the product during this purification step. The oil was further dried by stirring at room temperature under vacuum for three hours before it was stored at −21 °C under argon in a glove box.

1H NMR (300 MHz, CDCl3) δ = 8.17 (d, J = 8.0 Hz, 1H), 7.95–7.81 (m, 2H), 7.63–7.42 (m, 4H), 5.07 (q, J = 12.0 Hz, 2H), 3.85 (dt, J = 11.4, 3.3 Hz, 1H), 3.53 (dd, J = 11.5, 5.8 Hz, 1H), 3.26–3.19 (m, 1H), 2.84–2.79 (m, 1H), 2.65 (dd, J = 5.0, 2.7 Hz, 1H) ppm.

Synthesis of the allyl-PEO-OH macroinitiator

The synthesis of the allyl-PEO-OH macroinitiator was carried out in a BüchiGlas Uster Picoclave. First, allyl alcohol (205 μL, 0.003 mmol) was dissolved in 110 mL of dry THF in a GL45 bottle under argon in a glove box. Allyl alcohol was deprotonated by dropwise addition of a filtrated solution of diphenylmethyl potassium (DPMK) in THF until the color of the solution remained slightly yellow. The bottle was then closed and the solution was transferred to the reactor via the PTFE tubes attached under argon. The solution was then heated to 45 °C under stirring before 16 g of distilled ethylene oxide (EO) were added from the glass/stainless steel burette used for EO storage via a mass flow controller within 45 minutes. Stirring was continued for 23 hours. Subsequently, the reaction was quenched by addition of 2 mL of EtOH. The amount of solvent was reduced under reduced pressure. The crude allyl-PEO-OH macroinitiator was then purified by precipitation into cold diethyl ether from concentrated THF solution (2×) and further dried by azeotropic distillation of water and solvent residues with toluene (3×) and drying at 90 °C under high vacuum. The polymer was stored at room temperature under argon in a glove box. The preparation of the DPMK solution as well as a detailed description of the reactor used for ethylene oxide polymerization is given elsewhere.40

Allyl-PEO-OH: 1H NMR (300 MHz, CDCl3) δ = 6.00–5.81 (m, 1H), 5.25 (d, J = 17.2 Hz, 1H), 5.15 (d, J = 11.7 Hz, 1H), 4.00 (d, J = 5.6 Hz, 2H), 3.62 (s, 361H).

Synthesis of the diblock terpolymers

In a typical reaction, dry polyethylene oxide (allyl-PEO-OH, 4.000 g mol−1) was dissolved in THF in a microwave vial in a glove box at a concentration of 30 mg mL−1. Subsequently, a small excess of dried, solid potassium hydride was added, the vial was sealed and the suspension was stirred at 70 °C for one hour. Then, a 90[thin space (1/6-em)]:[thin space (1/6-em)]10 wt% mixture of the chosen hydrophobic glycidyl ether monomer and FGE was added. The mixture was allowed to stir at 70 °C for 24 hours. The polymerization was then quenched by adding 0.1 mL of dry methanol or ethanol. The solvent was evaporated under reduced pressure and the resulting diblock terpolymers were dried under vacuum.

For purification of the diblock terpolymers containing hydrophobic monomer 1 and 2, 100 mg of “crude” diblock terpolymer were adsorbed onto 1 g of silica gel. The dried gel was transferred to a glass frit and washed with 80 mL of a 5[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture of iso-propanol and dichloromethane before eluting the product with 50 mL of a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture of dichloromethane and methanol. The solvent of the second phase was evaporated under reduced pressure and the diblock terpolymer was dried under high vacuum.

The diblock terpolymers containing hydrophobic monomer 3 and 4 as well as all diblock terpolymers with a DP below 10 were dissolved in a small amount of dichloromethane and purified via precipitation in cold diethyl ether. The precipitate was dried under high vacuum at 90 °C for several hours.

PEO-b-P(EHGE-co-FGE): 1H NMR (300 MHz, CDCl3) δ = 7.35 (s), 6.27 (s), 6.00–5.77 (m), 5.25–5.11 (m), 4.40 (s), 4.00 (d, J = 5.8 Hz), 3.91–3.09 (m), 1.52–1.39 (m), 1.39–1.11 (m), 0.97–0.73 (m) ppm.

PEO-b-P(HHGGE-co-FGE): 1H NMR (300 MHz, CDCl3) δ = 7.36 (s), 6.28 (s), 6.00–5.81 (m), 5.30–5.12 (m), 4.43 (s), 4.00 (d, J = 5.7 Hz, 1H), 3.70–3.35 (m), 1.65–1.40 (m), 1.37–1.18 (m), 0.86 (t, J = 6.4 Hz) ppm.

PEO-b-P(NGE-co-FGE): 1H NMR (300 MHz, CDCl3) δ = 7.96 (s), 7.85–7.60 (m), 7.55–7.20 (m), 6.16 (s), 6.04–5.84 (m), 5.35–5.15 (m), 4.77 (s), 4.26 (s), 4.04 (d, J = 5.7 Hz, 1H), 3.95–3.30 (m) ppm.

PEO-b-P(BGE-co-FGE): 1H NMR (300 MHz, CDCl3) δ = 7.46–7.14 (m), 6.24 (s), 6.02–5.82 (m), 5.36–5.15 (m), 4.64–4.42 (m), 4.38 (s), 4.04 (d, J = 5.7 Hz), 3.98–3.31 (m) ppm.

Micelle preparation

For micelle formation, 5 mg of the desired diblock terpolymer were dissolved in THF in a glass vial. The solvent was evaporated slowly under reduced pressure in order to form a diblock terpolymer film in the vial. Then, the diblock terpolymer film was directly dissolved in 5 mL of micropure water under stirring for three days, resulting in a micellar solution containing 1 mg mL−1 diblock terpolymer.

For the preparation of core-crosslinked micelles, 5 mg of the desired diblock terpolymer was dissolved in 0.5 mL of THF. 1,1′-(Methylenedi-4,1-phenylene)bismaleimide were dissolved in THF (1 mg mL−1) and the desired amount of bismaleimide compared to the amount of furfuryl units present in the diblock terpolymers (one equivalent of maleimide units compared to the amount of furfuryl units present in the diblock terpolymer) was added as solution. The solution was homogenized and transferred to a glass vial. The solvent was evaporated slowly under reduced pressure in order to form a diblock terpolymer/crosslinker film in the vial. Although we cannot fully exclude that already some Diels–Alder reactions occur during this period, we assume mainly monoaddition to happen in this case as subsequent direct dissolution in 5 mL of micropure water under stirring for two days was possible. Afterwards, the vials were sealed and stirred at 60 °C for further 24 hours. The vials were allowed to cool to room temperature and the concentration was adjusted to 1 mg mL−1 by refilling the vial with micropure water prior to further investigation if necessary.

Control samples of non-crosslinked micelles used for comparison were also stirred for two days to allow the polymer film to dissolve and subsequently heated to 60 °C for 24 hours.

For the investigation of the influence of ultrasound on micellar dimension and morphology, 2 mL of a micellar solution of crosslinked and non-crosslinked micelles, respectively, were treated with 1 s ultrasound pulses for different times using an ultrasonication finger with an amplitude of 20%. The waiting time between two pulses was 1 s. The energy output monitored by the processor was 1 J for 1 × 1 s, 6 ± 1 J for 5 × 1 s, 12 ± 1 J for 10 × 1 s and 20 ± 2 J for 20 × 1 s. The samples were investigated via DLS within 30 minutes after the ultrasound treatment was finished.

Results and discussion

Synthesis and characterization of amphiphilic diblock terpolymers

To generate well-defined morphologies via self-assembly of diblock terpolymers in aqueous solutions, the underlying materials typically are required to be well-defined in terms of composition and dispersity. This is especially important if the generation of non-spherical morphologies within a rather narrow composition window in the corresponding phase diagram is of interest, as it is the case for polyether-based filomicelles. Additionally, the nature of the side chain can have a distinct influence on micellar morphologies, therefore the choice of the monomers plays a crucial role. We used commercially available as well as self-synthesized monomers with slight differences in sterical demand and possible monomer–monomer interactions in this study. The synthesis of these monomers and also the reasons behind comonomer choice will be discussed later. For the synthesis of such tailor-made poly(ethylene oxide)-based polymers as well as poly(glycidyl ethers) anionic ring opening polymerization typically is the method of choice, as this polymerization technique leads to narrow molar mass distributions and a comparably low amount of side reactions during the polymerization. A two-step approach was used for the synthesis of the diblock terpolymers in this study (Scheme 1): first, the PEO block was synthesized via AROP of ethylene oxide in a BüchiGlas reactor using allyl alcohol as initiator. Other alcohols, such as 2-methoxyethanolate to generate mPEO, can also be applied as initiators.18 In this case, allyl alcohol was chosen in order to introduce an end group to the hydrophilic block that can be functionalized afterwards. As the reaction was quenched using ethanol as protic reagent, a terminal hydroxyl group is introduced and the resulting allyl-PEO-OH was purified via precipitation in cold diethyl ether and residual traces of water were removed via azeotropic distillation with toluene. A molar mass of 4000 g mol−1 and a narrow dispersity of 1.09 were found in SEC measurements after purification. For the attachment of the second, hydrophobic block, the allyl-PEO-OH macroinitiator was dissolved in THF under argon in a glove box and deprotonated using solid KH at 70 °C. Then, a mixture of 90 wt% of the desired hydrophobic monomer and 10 wt% of FGE as crosslinkable comonomer were added. The polymerizations were quenched using methanol or ethanol after 24 hours.
image file: c9py01054h-s1.tif
Scheme 1 Synthesis of the allyl-PEO-OH macroinitiator and subsequent copolymerization of hydrophobic glycidyl ethers and FGE via anionic ring opening polymerization to obtain amphiphilic diblock terpolymer polyethers.

The crude diblock terpolymers were purified using two different methods for the removal of residual monomer and low molecular weight polymeric species formed due to transfer reactions caused by proton abstraction in α-position to the epoxide ring by growing polymer chain ends.26 For the diblock terpolymers containing aliphatic branched side groups, the crude products were adsorbed onto silica gel, residual monomer and byproducts were removed by washing with iso-propanol/DCM (5[thin space (1/6-em)]:[thin space (1/6-em)]1), and the purified product was desorbed from silica gel using a mixture of DCM and MeOH (1[thin space (1/6-em)]:[thin space (1/6-em)]1). For diblock terpolymers containing aromatic side groups and diblock terpolymers with hydrophobic blocks with DPs below 10, precipitation of the crude product in cold diethyl ether resulted in removal of both hydrophobic byproducts and residual monomer.

Different diblock terpolymers containing four different hydrophobic monomeric species, respectively, with molar masses between 5500 g mol−1 and 9000 g mol−1 and narrow dispersities between 1.06 and 1.18 were obtained. The compositions were calculated from the 1H NMR spectra by comparing the PEO signal and the side chain signals of the hydrophobic block. Molar masses, dispersities, and compositions of all diblock terpolymers are listed in Table 1, the results of SEC and 1H NMR analyses are presented in Fig. S1–S3.

Table 1 Overview of characterization data for diblock terpolymers and the corresponding micelles, the hydrodynamic radii were obtained in aqueous solution (1 mg mL−1). The subscript denotes the weight percentage of the hydrophilic and hydrophobic block, respectively
  M n (NMRa) g mol−1 M n (SECb) g mol−1 Đ Hydrophobic blocka wt% Crosslinkable comonomera RHz,app nmc Radius r nmd Morphology
Wt% Units (ca.)
a 1H NMR, 300 MHz, CDCl3. b SEC, THF, PEO calibration. c Determined by DLS, distribution with the lowest hydrodynamic radius in intensity-weighted CONTIN plots. d Determined by cryo-TEM evaluating at least 40 individual micelles. For worms and vesicles, d/2 of the worm-like aggregate/vesicle wall is listed here.
Allyl-PEO-OH 4000 1.09
PEO69-b-P(EHGE-co-FGE)31 5800 8700 1.06 31 3 1 6.1 4.6 ± 0.8 Spheres/worms
PEO54-b-P(EHGE-co-FGE)46 7500 10[thin space (1/6-em)]000 1.06 46 4 2 4.3 6.3 ± 1.1 Worms
PEO48-b-P(EHGE-co-FGE)52 8300 10[thin space (1/6-em)]900 1.06 52 5 3 8.7 5.5 ± 0.8 Worms
PEO44-b-P(EHGE-co-FGE)56 9000 11[thin space (1/6-em)]000 1.16 56 10 6 5.0 6.2 ± 0.7 Worms
PEO73-b-P(HHGGE-co-FGE)27 5500 7900 1.07 27 3 1 6.2 4.5 ± 0.6 Spheres
PEO54-b-P(HHGGE-co-FGE)46 7500 10[thin space (1/6-em)]200 1.08 46 9 4 5.1 4.4 ± 0.6 Worms
PEO49-b-P(HHGGE-co-FGE)51 8300 10[thin space (1/6-em)]900 1.08 51 4 2 7.6 5.6 ± 1.0 Worms/vesicles
PEO45-b-P(HHGGE-co-FGE)55 8800 9700 1.08 55 5 3 8.5 6.4 ± 0.9 Worms/vesicles
PEO62-b-P(NGE-co-FGE)38 7100 8700 1.18 38 6 3 6.9 5.1 ± 0.8 Worms
PEO59-b-P(BGE-co-FGE)41 7500 9700 1.13 41 6 3 6.6 5.1 ± 0.9 Spheres/worms


The four different monomeric species that were used in the syntheses of the diblock terpolymers investigated in this study are displayed in Fig. 1 and can be separated into two groups: branched aliphatic (1 & 2) and aromatic side chains (3 & 4).


image file: c9py01054h-f1.tif
Fig. 1 Different hydrophobic glycidyl ethers used for the synthesis of the diblock terpolymers investigated in this study.

The majority of studies on the self-assembly of polyether-based AB diblock copolymers is dealing with hydrophobic monomers of lower molar mass and sterical demand. Nevertheless, examples highlighting the advantages of larger, complex side groups for directing the self-assembly of such materials can be found in literature.4 For example, Majdanski et al. showed that worm-like aggregates are readily accessible if the commercially available, branched monomer EHGE (1) is used for the formation of the hydrophobic block of polyether-based AB diblock copolymers.18

To investigate the influence of the branched structure on micellar morphologies formed in aqueous solution, another branched hydrophobic monomer (HHGGE, 2) with higher molar mass and sterically more demanding side chains was synthesized (Scheme 2). In a first step, solketal was converted into the corresponding allyl ether using allyl chloride and solid KOH in hexane as base.41 After acidic removal of the isopropylidene protective group, both hydroxyl groups were etherified with 1-bromohexane.42 Subsequently, the allyl group was epoxidized using mCPBA.43 Both branched comonomers were used in polymerizations and the resulting diblock terpolymers were investigated concerning their self-assembly in aqueous solution.


image file: c9py01054h-s2.tif
Scheme 2 Synthesis of the branched hydrophobic glycidyl ether HHGGE.

Besides sterical effects, also non-covalent interactions between different groups in the side chain might play a crucial role in influencing solution structures of polyether-based AB diblock copolymers. Therefore, not only aliphatic, but also aromatic comonomers (3 & 4) were used. It has been reported that π–π-interactions are able to significantly influence the self-assembly of polymeric materials towards larger, more complex structures.44 Hence, the substitution of aliphatic with aromatic side groups might lead to similar results as increasing the sterical demand of the hydrophobic block. The effect of π–π-stacking can be enhanced by solvophobic effects, leading to a larger tendency of aromatic moieties to form π-stacks to avoid unfavorable contact to the surrounding medium.45 Both effects might influence the self-assembly of flexible, polyaromatic structures in selective solvents. Two aromatic comonomers were investigated in this study, BGE (4) and NGE (3). Here, especially the comparison of the diblock terpolymers containing different aromatic monomers directly as well as to their aliphatic counterparts with comparable composition was of interest. Therefore, no investigations on the variation of the DP of the hydrophobic block were made during this study. While BGE was commercially available, NGE had to be synthesized (Scheme 3). Here, naphthalene was bromomethylated using a slightly modified procedure as described by van der Made et al.46 Then, the corresponding allyl ether was formed by deprotonation of allyl alcohol with NaH and addition of the bromide,47 followed by epoxidation of the double bond using mCPBA.43


image file: c9py01054h-s3.tif
Scheme 3 Synthesis of NGE (3) as hydrophobic comonomer.

Besides sterical and electronic properties of the monomers within the hydrophobic block also the overall block length ratio governs self-assembly and the resulting micellar morphologies. Therefore, different block length ratios were targeted by using a constant length of the PEO segment and varying molar masses of the hydrophobic block,48 ranging from 27 wt% (PEO73-b-P(HHGGE-co-FGE)27) to 56 wt% (PEO44-b-P(EHGE-co-FGE)56). A weight fraction of 40 to 50 wt%, depending on the chosen comonomer, seemed to be the most promising range for the generation of worm-like structures and the investigation of transitions in the phase diagram proceeding from the worm-like phase. Keeping the molecular weight of the hydrophilic block constant allows to start the block extension of PEO from exactly the same macroinitiator, thereby excluding morphological changes due to slight variations in Mn or Đ of the hydrophilic segment.

Micellization of amphiphilic diblock terpolymers

Micellization was carried out by directly dissolving the diblock terpolymers in micropure water at a concentration of 1 mg mL−1. To ensure complete dissolution, the initial diblock terpolymer/water systems were stirred for three days. Slightly turbid solutions were obtained, and no sedimentation was observable if stirring was stopped even after several weeks. Comparison to the conventional solvent switch method, which involves dissolving the diblock copolymer in a non-selective solvent, followed by subsequent transfer to a selective solvent, revealed differences in the morphologies formed (see also discussion later for specific examples). Nevertheless, direct dissolution was used for the studies presented here. The micellar solutions were afterwards investigated via DLS and cryo-TEM measurements, revealing insights on the influence of the different hydrophobic comonomers and the lengths of the hydrophobic block chosen on the resulting micellar structures (Fig. S4,Fig. 2–4, Table 1). Please note that hydrodynamic radii calculated via CONTIN analysis of DLS measurements are calculated based on the assumption of a spherical particle and, therefore, are not directly applicable, as different translational and rotational diffusion coefficients found for non-spherical particles lead to the presence of several hydrodynamic radii distributions. Especially for anisotropic nanostructures, dimensions obtained from cryo-TEM micrographs provide more reliable data, even if it is only possible to analyze small sample partitions using this method. Additionally, due to a lack of contrast between the PEO corona and the background, the cross-sectional radius obtained here generally refers to the micellar core. Nevertheless, the size distribution referring to the lowest hydrodynamic radius in the intensity-weighted CONTIN plots often is in good agreement with the radius of spherical micelles or the cross-sectional radius of worm-like structures found in cryo-TEM micrographs. One possible reason for the latter might be the high flexibility of our system even in aqueous environment as described later in this paragraph. Therefore, this value is given for 〈RHz,app in Table 1.
image file: c9py01054h-f2.tif
Fig. 2 Cryo-TEM micrographs of micellar solutions (1 mg mL−1) of PEO73-b-P(HHGGE-co-FGE)27 (A), PEO54-b-P(HHGGE-co-FGE)46 (B) and PEO49-b-P(HHGGE-co-FGE)51 (C). A change in morphology from spheres over worm-like structures to a vesicle/worm mixed phase is visible. The main hydrophobic side group as well as the weight fraction of the hydrophobic block are depicted together with the corresponding structures.

image file: c9py01054h-f3.tif
Fig. 3 Cryo-TEM micrographs of micellar solutions (1 mg mL−1) of PEO69-b-P(EHGE-co-FGE)31 (A) and PEO44-b-P(EHGE-co-FGE)56 (B). A change in morphology from a sphere/worm mixed phase to long, worm-like micelles can be observed. The main hydrophobic side group as well as the weight fraction of the hydrophobic block are depicted together with the corresponding structures.

image file: c9py01054h-f4.tif
Fig. 4 Cryo-TEM micrographs of micellar solutions (1 mg mL−1) of PEO62-b-P(NGE-co-FGE)38 (A) and PEO59-b-P(BGE-co-FGE)41 (B). While for PEO62-b-P(NGE-co-FGE)38, long worm-like structures comparable to the structures formed from PEO-b-P(EHGE-co-FGE) diblock terpolymers are visible, PEO59-b-P(BGE-co-FGE)41 aggregates form a mixed phase of spheres and worm-like structures.

Furthermore, cryo-TEM micrographs show that the micellar morphologies formed depend not only on the main hydrophobic comonomer, but also on the weight fraction of the hydrophobic block. Mainly worm-like structures of varying length were obtained for the block length ratios investigated here. In case of PEO-b-P(EHGE-co-FGE) and PEO-b-P(HHGGE-co-FGE) also spheres or spheres and short filomicelles as a mixed phase can be observed for lower weight fractions of the hydrophobic segment, indicating the boundaries of the worm-like phase. For PEO-b-P(HHGGE-co-FGE), also the upper limit for the formation of worm-like structures was found, as large vesicles were observed upon using weight fractions of around 50%. In contrast to that, PEO-b-P(EHGE-co-FGE) seems to exhibit a rather broad window for worm-like micelles, as this phase is still present at 56 wt% of the hydrophobic block. As an example for aromatic comonomers, PEO-b-P(NGE-co-FGE) exhibited worm-like nanostructures at a lower hydrophobic block ratio if compared to PEO-b-P(BGE-co-FGE). Nevertheless, in all cases more compositions would have to be investigated to allow for a reliable and quantitative assessment of phase boundaries. A representative overview over all cryo-TEM samples can be found in the ESI (Fig. S5).

Please also note that using the solvent switch method (exemplarily for PEO-b-P(EHGE-co-FGE)) by dissolving the diblock terpolymer in THF first, followed by the addition of micropure water and evaporation of THF, distinctly shorter worm-like micelles together with spherical structures were generated (Fig. S6). This might indicate that THF as non-selective solvent was not removed completely from the micellar core by stirring the solutions in open vials for several days and leads to a slightly different environment and a local improvement of the solvent quality for the core-forming block. Also, direct addition of water to THF solutions of the respective diblock terpolymers led to comparable results.49 As another interesting note, mixing of micellar solutions containing spherical micelles (formed from PEO50-b-P(tBGE-co-FGE)50 synthesized in former studies, see ESI, Fig. S7) and worm-like micelles (PEO48-b-P(EHGE-co-FGE)52, Fig. S7) both formed by direct dissolution, followed by stirring for three days does not lead to a mixed phase, but to apparently only worm-like micelles, indicating that the micellar systems are capable to rearrange to a certain extent, even in aqueous media.48 This might be explained by the fact that the direct dissolution method often generates unimers in aqueous solution and also the core-forming polymer block is flexible and exhibits low Tg. Besides morphological aspects, the direct dissolution method additionally excludes any influence of remaining non-selective solvent.50

In some cases, a regular alignment of the worm-like micelles was observable in cryo-TEM micrographs. This might be a result of confinement within the vitrified ice film, but it provides a hint on the thickness of the micellar corona of the worm-like aggregates.51 For PEO44-b-P(FGE-co-EHGE)56, in addition to the radius of the micellar core with 6.2 ± 0.7 nm, an approximate corona thickness of 6.3 ± 0.5 nm would be determined if PEO hydration and potential corona interpenetration effects are neglected, leading to an approximated overall cross-sectional diameter of 25 nm for these structures. This would result in significantly larger values than the ones obtained from DLS measurements. Hydration of the PEO chains in the micellar corona might be a reason for that, which might in contrast also influence the values detected via DLS.

Comparing the results obtained here with PEO-b-P(tBGE-co-FGE) diblock terpolymer micelles reported previously,16 the use of branched aliphatic comonomers in general leads to the formation of more complex solution structures, even if the molar mass of the hydrophobic block is lower. If the critical packing parameter (CPP) model is applied, it can be stated that the replacement of tBGE with the same weight fraction of HHGGE or EHGE in a diblock terpolymer apparently leads to a higher CPP value. While, for example, spherical micelles were obtained from PEO25-b-P(tBGE-co-FGE)75 in earlier studies, PEO-b-P(HHGGE-co-FGE) diblock terpolymers only form small spherical micelles with a radius of 4.5 ± 0.6 nm at very low molar masses of the hydrophobic block, for example for PEO73-b-P(HHGGE-co-FGE)27 (Fig. 2A). PEO54-b-P(HHGGE-co-FGE)46 diblock terpolymers already form long worm-like structures (Fig. 2B). While the worm diameter is very homogeneous with around 9 nm, varying worm lengths ranging from 100 nm to >1 μm can be found. For higher molar masses of the second block, mixed phases consisting of worms and polydisperse vesicles can be observed (Fig. 3C). Cross-sectional diameters of the worm-like micelles slightly increase with increasing block length of the hydrophobic block (5.6 ± 1.0 nm for 51 wt%, 6.4 ± 0.9 nm for 55%) while worm lengths do not change significantly. The vesicles formed show a shell thickness comparable to the worm diameter and are rather polydisperse in size.

A similar behavior can be observed for micelles formed by PEO-b-P(EHGE-co-FGE). Here, already a mixed fraction of spheres with a radius of 4.6 ± 0.8 nm and worm-like micelles of different length was observed for the lowest molar mass of the hydrophobic block investigated, PEO69-b-P(EHGE-co-FGE)31 (Fig. 3A). Even if structures with lengths >1 μm can already be found here, filomicelles of intermediate lengths from 100 to 300 nm are observed more frequently. Also, in this case a significantly larger hydrodynamic radius would be expected compared to the cross-sectional radius obtained from cryo-TEM. For increasing weight fractions of the hydrophobic block between 46 and 55 wt%, mainly long, worm-like structures were observed (Fig. 3B). In contrast to the PEO-b-P(HHGGE-co-FGE) samples, no clear increase in the worm diameter with increasing hydrophobic block length was observed (6.3 ± 1.1 nm at 42%, 5.5 ± 0.8 nm at 52 wt% and 6.2 ± 0.7 nm at 56 wt%), but this might also be due to a differing contrast in the cryo-TEM sample of PEO58-b-P(EHGE-co-FGE)42. The fact that worm-like structures are already present at a lower molar mass of the hydrophobic block compared to PEO-b-P(HHGGE-co-FGE) might be a result of a slight variation in composition (69 to 73 wt% hydrophilic block content), but also the sterical demand of EHGE in comparison to HHGGE differs. In combination with a change in polarity (ether groups in the side chain compared to alkyl chains), this might explain the different weight fractions where a transition to mixed worm/vesicle phases is observed. Especially the latter might lead to a different packing of EHGE chains within the micellar core and presumably to a broader composition window for worm-like aggregates in the corresponding phase diagram.

Comparing aliphatic and aromatic side groups with regard to molar mass and sterical demand in general is difficult, as not only different shape and flexibility, but also electronic effects in case of the aromatic systems have to be taken into account. First, it can be mentioned that the two diblock terpolymer species investigated here containing aromatic side groups also show a tendency to form non-spherical aggregates at lower molar masses of the hydrophobic block compared to earlier investigated tert-butyl side groups.16 Further, it was found that at a similar molar mass of the hydrophobic block, the diblock terpolymer containing aromatic side chains with a higher sterical demand (NGE) was more prone to form worm-like structures (Fig. 5A). The filomicelles obtained show a cross-sectional radius of 5.1 ± 0.8 nm and lengths up to 1 μm, but shorter fragments of around 100 to 500 nm are dominating. The sample containing BGE units formed a mixed fraction of spheres of similar size (5.1 ± 0.7 nm) and rather short worms with a cross-sectional diameter comparable to that of the spheres (Fig. 5B). The length of the micelles is especially inhomogeneous here, as short fragments below 100 nm, long filomicelles of around 1 μm and structures of intermediate lengths can be found in equal amounts.


image file: c9py01054h-f5.tif
Fig. 5 Cryo-TEM micrographs of aqueous micellar solutions (1 mg mL−1 diblock terpolymer) of PEO44-b-P(EHGE-co-FGE)56 (A) without and (B) with crosslinker added after three days of stirring at room temperature and another 24 h of stirring at 60 °C (crosslinking).

In general, the data shown here does not yet allow to determine to which extent π–π-stacking contributes to the formation of anisotropic micellar morphologies from polyether block copolymers. Nevertheless, as the aromatic side groups are densely packed within the micellar core, an energetically favorable arrangement of the side chains seems at least probable.

Core crosslinking of worm-like micelles

In the next step, the generated micelles were core-crosslinked by reacting the furfuryl units incorporated in the micellar core with an encapsulated bifunctional crosslinker molecule. Here, bismaleimides are suitable, as they undergo Diels–Alder reactions with the furfuryl units at moderate temperatures (60 °C).52 This technique was already applied and investigated for spherical polyether-based micelles generated via the solvent switch method. In this case, the bismaleimide was incorporated into the micellar core by dissolving the block copolymer and the crosslinker together in a suitable solvent, e.g. THF, and adding this solution dropwise to micropure water. Self-assembly as well as evaporation of THF was allowed to proceed for 24 hours before the samples were heated to induce crosslinking. In this study, the crosslinking procedure was transferred to worm-like systems generated via direct dissolution.15,16 Here, a diblock terpolymer/crosslinker film was generated in the first step. The diblock terpolymer and 1,1′-(methylenedi-4,1-phenylene)bismaleimide as crosslinker were dissolved in THF and transferred to a glass vial. The solvent was then slowly evaporated under reduced pressure to form a film and guarantee the absence of organic solvent during the subsequent micellization process. SAXS analysis indicated phase separation at this stage and the formation of a lamellar structure with a domain size of ca. 40 nm (Fig. S8). Afterwards, the film was directly dissolved in micropure water and the samples were stirred for three days before crosslinking was carried out by heating the samples to 60 °C for one day. Crosslinked and non-crosslinked samples used in the study were treated analogously.

Suitable amounts of crosslinker were determined in our former studies by quantitative analysis of the crosslinked FGE moieties present in the polymeric micelles using HR-MAS 1H NMR spectroscopy of samples swollen in CDCl3. For this study, one equivalent of crosslinker per two equivalents of crosslinkable furfuryl unit in the diblock terpolymer was used. Comparison of HR-MAS 1H NMR spectra of crosslinked and non-crosslinked micelles generated from PEO44-b-P(EHGE-co-FGE)56 shows almost complete disappearance of the 1H NMR signals of the furfuryl units and the formation of crosslinked species (Fig. S9), proving the application of a suitable amount of crosslinker as well as successful crosslinking in general. Integration of the 1H NMR signals and normalization using the signals of the ethylhexyl group results in a degree of crosslinking of approximately 80%.

In contrast to earlier work, preservation of the micellar morphology upon encapsulation of a crosslinker molecule depicts an additional challenge for worm-like systems, as the window for this morphology in the phase diagram is often extremely narrow.3,53 On the other hand, crosslinking of non-spherical structures might also play a completely different role, as not only disassembly of the structures upon dilution or a change in the surrounding medium can be prevented, but also morphological changes can be suppressed. It might even be possible to separate crosslinked worm-like and spherical micelles, for example by ultracentrifugation.54 In this way, pure and long-term stabilized samples of worm-like micelles can be obtained from different diblock terpolymer species, opening up a larger library of polymers that can be used for the investigation of quantitative structure–property relationships in such materials. Few examples for crosslinked worm-like solution structures are also already reported in literature. Often, crosslinking is carried out via free radical polymerization of allyl groups present in the aggregates, but also the usage of small-molecule crosslinkers, for example dihydrazides for crosslinking of ketone functionalities, is reported.53,55,56 The crosslinking process, which is generally applicable to all of the solution structures formed from the diblock terpolymers presented here, is exemplified for PEO44-b-P(EHGE-co-FGE)56 in this paragraph.

A direct comparison of cryo-TEM micrographs of crosslinked and non-crosslinked samples in case of PEO44-b-P(EHGE-co-FGE)56 showed some additional branching after crosslinking (Fig. 5). Also in DLS data, a slight tailing towards higher values of τ was observable (Fig. S10), which might be caused by additional branching points. Nevertheless, this can also be related to small fractions of the crosslinker aggregating and precipitating from solution. We therefore assume that both the encapsulation and the subsequent crosslinking do not significantly affect the micellar morphology in the presented cases.

To further prove successful crosslinking, both crosslinked and non-crosslinked samples were diluted with four parts of a non-selective solvent for both blocks. In both cases, the count rates in DLS experiments decreased after addition of the non-selective solvent, but the decrease was more pronounced for non-crosslinked samples. Furthermore, whereas a similar size distribution pattern was observable in CONTIN plots of crosslinked samples in 4[thin space (1/6-em)]:[thin space (1/6-em)]1 1,4-dioxane/water or 4[thin space (1/6-em)]:[thin space (1/6-em)]1 THF/water mixtures, only a broad distribution at lower hydrodynamic radii was visible for non-crosslinked samples, indicating dissolution of the micelles and the presence of diblock terpolymer unimers (Fig. S10B). The same was observable for the crosslinked micelles with very low DPs of the hydrophobic block and therefore also a very low content of FGE units, indicating that a certain number of FGE units per chain is necessary to achieve efficient core crosslinking.

Morphological changes in worm-like micelles by ultrasonication

As worm-like structures with lengths >1 μm were often the dominating species in samples investigated here, we were interested in whether the length of these aggregates can be tuned, which would be a useful tool for later studies. Even if worm-like micelles can show an increased circulation time in the blood stream, cell uptake of overly long structures through cell membranes might not be possible on the other hand. Therefore, controlled generation of shorter fragments of filomicelles for drug delivery purposes would be of interest.29 To investigate the influence of ultrasonication on length and length distribution of worm-like micelles, both crosslinked and non-crosslinked micelles from PEO44-b-P(EHGE-co-FGE)56 were exposed to short ultrasonication pulses for different times. We chose this diblock terpolymer as model system, as it was shown that almost exclusively worm-like micelles are formed in aqueous solution upon direct dissolution. Crosslinked as well as non-crosslinked micelles were treated with 1 s ultrasound pulses for different overall exposure times and were subsequently investigated via DLS. First, it has to be mentioned that the transmission of the crosslinked, ultrasonicated micellar solutions decreased, while no changes were observable for non-crosslinked samples. Intensity-weighted CONTIN plots showed a decrease of the intensity and size of the hydrodynamic radius distribution below 10 nm (Fig. 6). A reason for that might be the formation of either unimers and possibly also larger aggregates from previously defined, worm-like structures. This effect was not visible in case of non-crosslinked micelles. One tentative explanation is that disassembly of the non-crosslinked structures during ultrasound treatment might occur as well, leading to a similar situation as during the initial direct dissolution. After the ultrasound treatment is stopped, the diblock terpolymers then re-assemble and form (similar) worm-like micellar aggregates.
image file: c9py01054h-f6.tif
Fig. 6 Intensity-weighted DLS CONTIN plots of crosslinked micelles (1 mg mL−1 diblock terpolymer) formed from PEO44-b-P(EHGE-co-FGE)56, treated with ultrasound for different times (A). (B) shows the enlarged plot from 1 to 10 nm. For 5 and more ultrasound pulses of 1 s, the signal assigned to the cross-sectional radius of the micelles disappears and a distribution around 2 to 3 nm appears, presumably referring to diblock terpolymer unimers.

Cryo-TEM investigations of the crosslinked micelles after ultrasound treatment revealed an increased amount of branched worm-like structures as well as the formation of toroids with additional branching points (Fig. 7A) in comparison to samples not exposed to ultrasonication (Fig. 5B). This is equivalent to the formation of the larger aggregates from defined worm-like micelles observed in DLS measurements. The structures remained intact for at least two weeks without further ultrasound treatment, indicating a certain kinetic stability of the formed aggregates (Fig. 7B). The low glass transition temperature (Tg) of the micellar core might be a reason for the irregular aggregation of the non-water soluble parts of the diblock terpolymer unimers and partially crosslinked micellar fragments produced by ultrasonication which are present afterwards. Even if the breakage of covalent bonds during ultrasonication treatment can be imagined, in our opinion the rearrangement of the crosslinked nanostructures originates from low crosslinking densities.


image file: c9py01054h-f7.tif
Fig. 7 Cryo-TEM micrographs of crosslinked micellar solutions (1 mg mL−1) of PEO44-b-P(EHGE-co-FGE)56 treated with 1 s ultrasound pulses for 5 s. Micrographs were taken directly after ultrasound treatment (A) and after two weeks at room temperature (B).

In case of block copolymers containing a higher Tg core-forming block, as used in most studies investigating the effect of ultrasonication on filomicelles, the crystallinity of the micellar core and not the presence of covalent bonds formed by crosslinking is the main reason for the stability of the micellar fragments, therefore re-assembly is also mainly driven by crystallization, leading to defined solution structures.57

Conclusion

Diblock terpolymers with a hydrophilic PEO block and a hydrophobic block containing 10% FGE as a crosslinkable comonomer as well as 90% of different hydrophobic glycidyl ether species were successfully synthesized and purified. The materials show narrow molar mass distributions and self-assembly via direct dissolution in micropure water resulted in different micellar morphologies starting from spherical micelles over worm-like structures to large vesicles as proven via DLS and cryo-TEM analysis. The morphology was dependent on the DP of the hydrophobic block as well as on the main hydrophobic monomer used. Mainly long worm-like structures were found for the compositions investigated in this study. Further, it was demonstrated that the preparation technique was crucial for their generation, as the conventional solvent switch method in different cases resulted in a mixed phase of spherical micelles and short worms, as it was observed for hydrophobic blocks of lower DP or with hydrophobic monomers of lower sterical demand. Additional crosslinking of the micellar core was carried out using a bismaleimide crosslinker and only minor structural differences between crosslinked and non-crosslinked samples such as additional branching points were found after crosslinking. Successful crosslinking was further proven via comparison of DLS measurements of crosslinked and non-crosslinked samples in a nonselective solvent mixture (THF/water 4[thin space (1/6-em)]:[thin space (1/6-em)]1) and swollen state HR-MAS 1H NMR measurements of micelles freeze-dried from aqueous solutions. Ultrasound treatment of the core-crosslinked micelles gave access to highly branched worm-like structures, presumably due to the (still) quite high chain mobility in the hydrophobic block.

The diblock terpolymers presented herein provide access to a small, but expandable library of crosslinkable glycidyl ether block copolymers featuring additional versatility concerning length, sterical demand, and possible monomer interactions within the side chain. Whereas micelle formation and core crosslinking seems to be rather straightforward, further work will focus on the influence of type and amount of crosslinker on core stability during ultrasound treatment of other suitable methods to control the length of worm-like micelles after self-assembly. This seems attractive, as length-tunable worm-like micelles with additional points for functionalization on the micellar surface (within the shell) are promising candidates for drug delivery applications.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors thank the German Research Council (DFG) for financial support of the project (SCHA 1640/16–1; GO 1100/4–1, and within the framework of the SFB1278 “PolyTarget”, projects A03 and C03), the Dutch Polymer Institute (DPI, technology area high-throughput-experimentation, project #690), U. S. Schubert and J. Vitz for the possibility and help during the polymerization of ethylene oxide, and Johannes B. Max for SAXS measurements. The cryo-TEM/TEM facilities of the Jena Center for Soft Matter (JCSM) were established with a grant from the German Research Council (DFG) and the European Fonds for Regional Development (EFRE). The HR-MAS-NMR facilities were supported by the DFG (INST 275/331-1 FUGG).

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Footnote

Electronic supplementary information (ESI) available: DLS correlation functions and additional DLS CONTIN plots. See DOI: 10.1039/c9py01054h

This journal is © The Royal Society of Chemistry 2019