LCST Behavior of Symmetrical PNiPAm-b-PEtOx-b-PNiPAm Triblock

Sep 28, 2016 - Hence, the binary mixture becomes cloudy while heating above the so-called cloud point temperature (Tcp).(7, 8). For this type of therm...
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LCST Behavior of Symmetrical PNiPAm‑b‑PEtOx‑b‑PNiPAm Triblock Copolymers Martin Sahn,†,‡ Turgay Yildirim,†,‡ Michael Dirauf,†,‡ Christine Weber,†,‡ Pelin Sungur,†,‡ Stephanie Hoeppener,†,‡ and Ulrich S. Schubert*,†,‡ †

Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstr. 10, 07743 Jena, Germany ‡ Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany S Supporting Information *

ABSTRACT: Poly(N-isopropylacrylamide) (PNiPAm) and poly(2ethyl-2-oxazoline) (PEtOx) represent two polymer types that are well-known for their lower critical solution temperature (LCST) behavior in aqueous media. To synthesize triblock copolymers containing both polymers, a crossover of two different polymerization methods was applied using a bifunctional initiator for the living cationic ring-opening polymerization (CROP) of EtOx. Quantitative end-functionalization with a trithiocarbonate resulted in a bifunctional PEtOx macro chain transfer agent (CTA). A series of well-defined PNiPAm-b-PEtOx-b-PNiPAm triblock copolymers were obtained by subsequent reversible addition−fragmentation chain transfer (RAFT) polymerization of NiPAm. The influence of the PNiPAm to PEtOx ratio on the thermoresponsive properties was intensively investigated via turbidimetry, dynamic light scattering, cryo transmission electron microscopy, and 1H NMR studies, revealing hydrogen bonds between both copolymer segments that strongly lower the phase separation temperature of aqueous solutions.

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INTRODUCTION Symmetrical triblock copolymers have long been applied as bulk materials due to the favorable properties that arise from phase-separated hard and soft blocks.1 More recent research is directed toward the application symmetrical triblock copolymers in solution, with a special focus on the incorporation of responsive blocks that can be used to alter the properties by an external trigger.2 Pluronics, i.e., triblock copolymers composed of poly(ethylene oxide) (PEO) and poly(propylene oxide), are the most well-known representatives of this type of copolymer, however, with a central thermoresponsive block.3 Among others, thermoresponsive blocks are of significant interest because the resulting materials exhibit non-Newtonian flow behavior or can be applied for the reversible formation of hydrogels.4−6 The latter is based on the collapse of a building block exhibiting lower critical solution temperature (LCST) behavior in aqueous solution.7,8 Among the variety of polymers that reveal this property, poly(N-isopropylamide) (PNiPAm) has remained the gold standard for 50 years because it “switches“ from a hydrophilic to a hydrophobic behavior slightly below body temperature at 32 °C.9 This transition is based on the formation of hydrogen bonds with the water molecules that keep the polymer in solution at low temperatures. Driven by entropy, these bonds are weakened at elevated temperature, leading to a collapse and an aggregation of the polymer chains.10 During this demixing process two phases with a higher and a lower polymer concentration are formed. Hence, the binary mixture becomes © XXXX American Chemical Society

cloudy while heating above the so-called cloud point temperature (Tcp).7,8 For this type of thermally induced sol−gel transition of aqueous ABA triblock copolymer solutions the central block has to be permanently hydrophilic. Therefore, PEO and PNiPAm have often been combined in different architectures.11−13 Solely relying on reversible addition−fragmentation chain transfer (RAFT) polymerization processes, similar properties have been achieved by utilization of a hydrophilic block based on vinylic monomers.4 Poly(2-ethyl-2-oxazoline) (PEtOx) represents another type of hydrophilic polymer that is known for its biocompatibility and stealth behavior and therefore has recently come into focus for biomedical applications.14−16 Poly(2-oxazoline)s (POx) with a propyl substituent reveal thermoresponsive properties close to body temperature.17 Hence, the thermally triggered micellization of diblock copolymers composed of PEtOx and poly(2-n-propyl-2-oxazoline)18 and poly(2-isopropyl-2-oxazoline)19 has been investigated in depth. POx can be obtained by applying a cationic ring-opening polymerization (CROP) mechanism.14,20 The livingness of this polymerization type enables the use of functional initiators and end-capping agents21 as well as the preparation of block copolymers by sequential monomer addition.22−24 A crossover of polymerization techniques is necessary to prepare block copolymers comprising PEtOx and Received: June 27, 2016 Revised: September 16, 2016

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DOI: 10.1021/acs.macromol.6b01371 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Representation of the Synthesis Route toward PNiPAm-b-PEtOx-b-PNiPAm Triblock Copolymers

PNiPAm building blocks. Besides the application of “click chemistry” approaches,25−28 functional initiators29 or end-capping agents30 as chain transfer agents (CTAs) for the RAFT polymerization process represent a reasonable synthetic alternative. The application of a bifunctional CROP initiator is a straightforward approach, minimizing the necessary synthetic steps to create a ABA triblock architecture with a central PEtOx block. As the central block is synthesized first and the two outer blocks are attached in a second synthetic step, the resulting triblock copolymer in fact reflects a BAB structure. Although this nomenclature is not applied consistently throughout the related literature,4−6 it easily depicts additional synthetic information and, hence, is used here. Allylic dihalides31,32 as well as aliphatic diiodides33 have been applied as bifunctional initiators but have rarely been used for the further preparation of triblock copolymers.34 Recently, the Tiller group reported a α,α′-dibromo-p-xylene (DBX) initiator suitable for the preparation of symmetrical BAB triblock copolymers that are solely based on poly(2-oxazoline)s via consecutive monomer addition.35,36 DBX represents a bifunctional initiator whose structure is based on the well-known CROP initiator benzyl bromide.37−40 However, the two positive partial charges are in close proximity, which can complicate a fast and simultaneous initiation of two POx chains, which is required for the synthesis of well-defined symmetrical triblock copolymers. By replacing the benze ring of DBX by a larger biphenyl spacer, we present a new bifunctional CROP initiator to prepare a hydrophilic middle block of a symmetrical BAB type triblock terpolymer (Scheme 1). End functionalization of the living ω-chain ends with a carboxylic acid-functionalized CTA30

resulted in a bifunctional macro chain transfer agent (MCTA) suitable to polymerize NiPAm via a subsequent RAFT polymerization process to obtain PNiPAm-b-PEtOx-b-PNiPAm triblock copolymers (P1 to P6 in Scheme 1). This approach enables the combination of two polymer types that cannot be obtained using a single polymerization technique avoiding a postpolymerization coupling of two individual blocks. The LCST behavior of the copolymers P1 to P6 was investigated in detail, since interaction of the two block types resulted in unexpected effects.

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EXPERIMENTAL PART

Materials. Unless specified otherwise, all solvents and compounds were used without further purification. 2-Ethyl-2-oxazoline (EtOx) was purchased by Acros Organics, dried over barium oxide, distilled, and stored under an argon atmosphere. N,N-Dimethylformamide (DMF, Sigma-Aldrich) was dried in a solvent purification system (Pure Solv EN, InnovativeTechnology) before use as polymerization solvent. Triethylamine (NEt3) was dried over potassium hydroxide and was distilled and stored under an argon atmosphere. 4,4′-Bis(bromomethyl)biphenyl (BBMBP, ≥97% purity), N-isopropylacrylamide (NiPAm, ≥97% purity), 4,4′-azobis(4-cyanovaleric acid) (ACVA, ≥98% purity), and sodium perchlorate (NaClO4) were purchased from Sigma-Aldrich. Lithium sulfate (Li2SO4) was obtained from Acros Organics. 2-(Butylthiocarbonothioylthio)propanoic acid (BTPA) was a kind gift from the BASF and used as received. Instruments. Size exclusion chromatography (SEC) was measured on a Shimadzu system equipped with a SCL-10A VP system controller, a LC-10AD VP pump, and a RID-10A refractive index detector using a solvent mixture containing chloroform, triethylamine, and isopropanol (94:4:2) at a flow rate of 1 mL min−1 on a PSS-SDV-linear S 5 μm column (PSS GmbH Mainz, Germany) at 40 °C. The system was calibrated with polystyrene (370 to 128 000 Da) standards. B

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utilizing a cryo stage. Images were acquired on a 1 × 1 k or a 4 × 4 k CCD camera. Synthesis. Kinetic Studies. A stock solution of EtOx (4 g, 40.35 mmol), BBMBP (686 mg, 2.01 mmol), and DMF (6.02 mL) with a [monomer] to [initiator] ratio ([M]/[I]) of 20 and a monomer concentration of 4 mol L−1 was prepared. Microwave vials were heated overnight at 110 °C and cooled down under argon. 1 mL aliquots of this stock solution were transferred to suitable microwave reaction vessels under an argon atmosphere, capped, and heated in an microwave synthesizer for a time period of 1, 2, 4, 6, 8, and 10 min. All samples were analyzed by means of SEC and 1H NMR spectroscopy. Synthesis of the Macro Chain Transfer Agent (MCTA). BBMBP (1.113 g, 3.27 mmol) and DMF (9.61 mL) were transferred to a preheated microwave vial under an argon atmosphere. After the initiator was dissolved completely, EtOx (6.433 g, 64.89 mmol) was added to the reaction vial. The closed vial was transferred to the autosampler of the microwave. After a prestirring period of 30 s, the solution was heated to a temperature of 140 °C for 12 min. The reaction vessel was cooled to room temperature by pressurized air. The living polymer chains were quenched by addition of BTBA (1.71 g, 7.21 mmol) in DMF (2 mL) through the septum of the vial. NEt3 (0.91 mL, 6.55 mmol) was added subsequently in the same manner. The endcapping process was carried out in an oil bath at 50 °C for 2 h. After cooling the reaction mixture to room temperature, chloroform (200 mL) was added. The organic phase was washed with saturated sodium bicarbonate solution (3× 100 mL) twice followed by a washing step with brine. The organic layers were dried over sodium sulfate, and the solvent was evaporated under reduced pressure. Finally the polymer was precipitated into diethyl ether (−20 °C), filtered, and dried in a vacuum oven overnight. MCTA (yield: 6.64 g) was obtained as a yellow solid. 1H NMR (300 MHz, CDCl3, the numbers indicate the assigned peaks in Figure 2): 7.55 (C−H (1)), 7.24 (C−H (1)), 4.80 (C−H (2)), 4.60 (CH2 (3)), 4.25 (CH2 (4)), 3.45 (CH2 (5)), 2.39 (CH2 (6)), 1.12 (CH3 (7)), 0.92 (CH3 (8)) ppm. DF = quant (99.3% determined by 1H NMR). Mn (MALDI) = 2200 g mol−1; Đ = 1.02; Mn (ESI) = 2760 g mol−1. General Procedure for the RAFT Polymerization of P1 to P6. For each polymerization, the [MCTA]/[ACVA] ratio was kept as 1/0.25 and the initial monomer concentration was 2 mol L−1 in DMF. In a representative example for P3, NiPAm (500 mg, 4.41 mmol), MCTA (196 mg, 0.074 mmol), and ACVA (5.16 mg, 0.018 mmol) were dissolved in DMF (2.20 mL) in a microwave vial. After taking a sample for the determination of the monomer conversion, the vial was closed with a suitable septum. The reaction mixture was subsequently flushed with argon for 1 h. The reaction vessel was heated in an oil bath at 70 °C for 16 h. The mixture was cooled to room temperature, and a sample for determination of monomer conversion was taken. The polymer was obtained by precipitation into cold diethyl ether. A second precipitation was performed using tert-butyl methyl ether to separate residual DMF. The polymer was dried in a vacuum oven overnight. 1H NMR (300 MHz, CDCl3, the numbers indicate the assigned peaks in Figure 5): 7.57 (C−H (1)), 7.23 (C−H (1)), 6.30 (NH (2)), 4.61 (CH2 (3)), 4.00 (CH (4)), 3.45 (CH2 (5)), 2.40 (CH2 (7)), 2.09 (CH2 (9)), 1.68 (CH (8)), 1.12 (CH3 (10)) ppm.

The polymerization of EtOx was performed in a Biotage Initiator Sixty microwave synthesizer. 1 H NMR spectra were recorded on a Bruker Avance 300 MHz and Bruker Avance 400 MHz using the residual solvent resonance as an internal standard. The chemical shifts are given in ppm relative to trimethylsilane. Matrix-assisted laser desorption/ionization time-offlight (MALDI-ToF) mass spectra were acquired with an Ultraflex III ToF/ToF instrument (Bruker Daltonics, Bremen, Germany). The instrument is equipped with a Nd:YAG laser. All spectra were measured in the positive reflector mode. The instrument was calibrated prior to each measurement with an external PMMA standard from PSS. For the sample preparation, separate solutions of polymer (10 mg mL−1 in chloroform), trans-2-[3-(4-tert-butylphenyl)-2-methyl2-propenylidene]malononitrile (DCTB) (30 mg mL −1 in chloroform), and doping of sodium iodide (NaI) (100 mg mL−1 in acetone) were prepared and mixed following the dried droplet spotting technique. 1 μL of the mixture was spotted onto the target plate. For the electrospray time-of-flight mass spectrometry (ESI-ToFMS) measurements, samples were analyzed by using a microToF Q-II (Bruker Daltonics) mass spectrometer equipped with an automatic syringe pump from KD Scientific for sample injection. The mass spectrometer was running at 4.5 kV, at a desolvation temperature of 180 °C, and was operated in the positive ion mode. Nitrogen was used as the nebulizer and drying gas. All fractions were injected using a constant flow rate (3 μL min−1) of sample solution. The instrument was calibrated in the m/z range from 50 to 3000 using a calibration standard (Tunemix solution) which is supplied from Agilent. All data were processed via Bruker Data Analysis software version 4.0. Cloud points were determined in a Crystal 16 from Avantium Technologies connected to a chiller (Julabo FP 40) at a wavelength of 500 nm. After sample preparation, all samples were stored in a fridge at 4 °C overnight to ensure the presence of homogeneous solutions. Prior to starting the measurement, all samples solutions were kept at 1 °C for 10 min inside the instrument. Subsequently, the solutions were heated and cooled at a rate of 1 K min−1 in a temperature range between 1 and 100 °C. Three consecutive heating/ cooling cycles were performed without interruption of the measurement using the heating program previously defined. If not specified otherwise, the cloud point temperature Tcp was defined as the temperature where the transmittance decreased to 50% in the second heating run. Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany) with a scattering angle of 173 °C. The samples were heated from 10 to 40 °C in 5 °C steps. At each step, the samples were equilibrated for 240 s, and then 3 × 30 runs were carried out. Each measurement was performed in triplicate. The intensity and the volume distribution of the particle size were calculated applying the nonlinear least-squares fitting mode. Cryo transmission electron microscopy (cryo-TEM) investigations were conducted on a FEI Tecnai G2 20 with an acceleration voltage of 200 kV. Samples (8.5 μL) were applied onto Quantifoil grids (Quantifoil, Germany, R2/2) utilizing a Vitrobot Mark IV virtrification system. Grids were transferred to the cryo-TEM holder (Gatan, USA)

Figure 1. Kinetic studies of the polymerization of EtOx initiated by BBMBP in DMF at 140 °C ([M]/[I] = 20, [M] = 4 mol L−1). Left: first-order kinetic plot of the polymerization (ln([M]0/[M]t) = [I]0kp,appt, [I]0 = 0.2 mol L−1, R2 = 0.991). Middle: Mn and Đ values obtained from SEC for the polymers of the kinetic study. Right: SEC traces of the polymers of the kinetic study (CHCl3/NEt3/isopropanol 94:4:2, RI detection, PS calibration). C

DOI: 10.1021/acs.macromol.6b01371 Macromolecules XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION

Cationic Ring-Opening Polymerization. The first step of the synthetic approach comprised the development of an efficient bifunctional initiating system for the CROP of EtOx that can provide an active cationic species at each end, which is required for the subsequent end functionalization with a CTA. Hence, BBMBP was investigated since both bromide functionalities are well separated from each other by the biphenyl spacer. The capability of BBMBP to act as an efficient initiator for the CROP of EtOx was investigated by performing kinetic studies in DMF at 140 °C using microwave irradiation as heating source. Exclusively unimodal molar mass distributions were obtained in the SEC elugrams (Figure 1, right), hinting toward the fact that both benzylic bromide moieties indeed initiated a growing POx chain. No bimodal molar mass distributions or shoulders were observed in the SEC traces, which would correspond to PEtOx chains initiated by only one benzyl bromide unit. The fact that the semilogarithmic kinetic plot (Figure 1, left) of the data derived from the kinetic studies is linear reveals the presence of a polymerization of pseudo first order. This shows that both initiating sites of the bifunctional initiator BBMBP are capable to initiate the CROP efficiently and quickly form oxazolinium species. As a result, the chain propagation is the step that determines the polymerization kinetics. From the slope of the plot, the polymerization rate constant kp,app can be extracted as kp,app = 0.03 L mol−1 s−1. However, it has to be taken into account that two cationic chains grow from one initiator moelcule simultaneously. Hence, the kp,app for the addition of monomers to one living chain corresponds to kp,app = 0.15 L mol−1 s−1. In addition, the molar mass of the PEtOx increases in a linear fashion with the monomer conversion and the dispersity values remain low even at high conversions (Đ ≤ 1.13, Figure 1, middle). Thus, the system of BBMBP in DMF at a temperature of 140 °C represents a suitable way to prepare PEtOx from a bifunctional initiator under excellent control. Functionalization of the Living Chain Ends. The next step included the functionalization of the living oxazolinium chain ends with a suitable CTA. This is in particular challenging since a quantitative attachment on both chain ends is required. Because of the living character of the CROP, an introduction of several functionalities to the ω-chain end using nucleophiles is possible. Quenching of the cationic oxazolinium species with a carboxylic acid in the presence of triethylamine leads to high degrees of functionalization.30,41 In a slight modification of established procedures, the reaction was performed for 2 h at

Figure 2. 1H NMR spectrum of MCTA (300 MHz, CDCl3) and assignment of the peaks to the schematic representation of the structure.

50 °C with 1.1 equiv of BTPA and an equimolar ratio of triethylamine per living polymer chain end. Thereby, the excess of carboxylic acid-functionalized CTA was kept at a minimum to avoid a cleavage of the trithiocarbonate, which would result in POx chains with a thiol end group. These would be prone to subsequent disulfide coupling reactions. Figure 2 shows the 1H NMR spectrum of the macro chain transfer agent (MCTA) and the structural assignment of the peaks. Signals derived from the initiator, the PEtOx chain, and the end groups introduced can be detected and integrated separately. This enables to calculate the DP. For this purpose, signal 3 in Figure 2, which corresponds to the methylene protons of the initiator, and the peak integrals assigned to the PEtOx chains were used (both peaks 5 and 6 are suitable). The resulting DP value of 20 corresponds well to the used [M]/[I] ratio. Furthermore, the quantitative degree of functionalization (DF) of the ω-chain ends with the CTA can be confirmed by comparison of the peak integrals derived from the initiator and the CTA, respectively (peaks 2 and 3 in Figure 2). Signal 4 can be assigned to the methylene protons adjacent to the ester species formed by the quenching step with the carboxylic acid. Hence, its presence represents a direct proof for the covalent attatchment of the CTA at the polymer. Also, the MALDI-ToF mass spectrum indicates the successful functionalization of the PEtOx on both chain ends (Figure 3). The m/z distance between two neighboring peaks corresponds to one EtOx repeating unit with a mass of 99 g mol−1. Only one main distribution is observed in the spectrum, which can be assigned to a sodiated species that carries two

Figure 3. MALDI-ToF mass spectrum of MCTA (DCTB, NaI). Left: full spectrum; center: zoomed area; right: calculated and measured isotopic pattern of the peak at m/z = 2262.1 [C8H13S3O2(C5H9NO)8C14H12(C5H9NO)8C8H13S3O2 + Na]+. D

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RAFT Polymerization. The synthesis of the triblock copolymers was attained via RAFT polymerization by utilization of the synthesized MCTA as a chain transfer agent and ACVA as initiator. A series of triblock copolymers was synthesized by variation of the ratio of MCTA and NiPAm to investigate the influence of the length of the outer segments on the LCST behavior of the aqueous solutions. The [MCTA]/[ACVA] ratio was kept constant as 1/0.25, and the monomer concentration was 2 mol L−1. Table 1 summarizes the characterization data of the resulting PNiPAm-b-PEtOx-b-PNiPAm triblock copolymers that were obtained by 1H NMR spectroscopy as well as by SEC. The SEC traces of the purified block copolymers and the MCTA are depicted in Figure 4. No considerable amounts of residual MCTA were evidenced by SEC measurements, which holds true even for the triblock copolymers measured directly from the reaction solution (see Figure SI 2). In addition, the shift to lower elution volumes and the increase of the molar mass throughout the triblock copolymer series P1 to P6 demonstrate the effectiveness of MCTA for the control of the molar mass during the RAFT polymerization. All molar mass distributions are narrow with Đ values below 1.25, which correspond to commonly reported dispersities for RAFT polymerizations. It has to be mentioned that the molar mass values obtained from the SEC measurements are based on a PS calibration and, hence, represent relative values. The composition of the purified polymers was determined by 1 H NMR spectroscopy using chloroform as solvent. As it is shown in Figure 5 (bottom), all characteristic signals of the PNiPAm and PEtOx blocks can be observed. The separated signal 4 includes two methylene protons of the PEtOx block (compare signal 4 in Figure 2) as well as the methine protons of the PNiPAm blocks. The signal labeled with 3 in Figure 5 corresponds to methylene protons of the PEtOx block. Hence, integration of these peaks enables to calculate the overall copolymer composition. Since the molar mass of the PEtOx block is known, also the molar mass of the final triblock copolymers P1 to P6 can be determined. As is evident from Table 1, the DP values of all PNiPAm blocks are slightly lower than expected from the used ratio of [MCTA]/[M] and the conversion (roughly 10−20%). P1 represents the only copolymer where the PNiPAm blocks are shorter than the central PEtOx block. For all other polymers P2 to P6, the PNiPAm-based blocks are longer than the PEtOx block, covering a wide range of DP’s. LCST Behavior of PNiPAm-b-PEtOx-b-PNiPAm Block Copolymers. A thermoresponsive behavior of the synthesized materials was expected since the outer PNiPAm blocks represent the polymer type most frequently investigated in

CTA end groups. A simulation of the isotopic pattern is in excellent agreement with the measured isotopic pattern of the investigated MCTA ionized with a sodium cation (see Figure 3, right). Moreover, no hydroxyl-terminated species can be observed, which underlines the complete functionalization of both living chain ends. A chain transfer reaction during the polymerization would result in a proton initiated species, which was detected neither. The ESI-ToF mass spectrum (Figure SI 1) shows a singly, a doubly, and a triply charged distribution. Each of those can be assigned to the desired MCTA structure that is ionized with one, two, or three sodium counterions, respectively. The isotopic pattern of every distribution corresponds well to the simulated isotopic pattern of the MCTA. Again, no hydroxylvterminated and proton-initiated species were observed. Since no molar mass discrimination effects are apparent from both mass spectra, the molar mass of MCTA was determined as well. Therefore, the ESI-ToF mass spectrum was deconvoluted prior to analysis. The resulting value of Mn = 2800 g mol−1 is in very good agreement with the molar mass determined from the 1 H NMR spectrum, while the molar mass value from MALDIToF mass spectrometry investigation is slightly lower (Mn = 2200 g mol−1). Also, the SEC trace of MCTA (see Figure 4)

Figure 4. Overlay of the normalized SEC traces of MCTA and the triblock copolymers P1 to P6 (CHCl3/NEt3/isopropanol 94:4:2, RI detection).

shows a narrow and monomodal molar mass distribution of the polymer chains with a dispersity of 1.15. As a result, it could be demonstrated that both living oxazolinium chain ends were functionalized quantitatively with a RAFT agent, making MCTA suitable for the preparation of symmetrical triblock copolymers.

Table 1. Characterization Data of the Synthesized MCTA and the Triblock Copolymers P1 to P6 sample

M/CTA

conva (%)

DP NiPAmb

DP NiPAmc

composition NiPAm−EtOx−NiPAm

Mn(NMR)c

Mn(SEC)d

Đd

MCTA P1 P2 P3 P4 P5 P6

0 20 40 60 80 100 120

96 97 97 96 97 97

0 20 39 58 77 97 116

0 16 32 54 64 84 98

0−20−0 8−20−8 16−20−16 27−20−27 32−20−32 42−20−42 49−20−49

2600 4500 6300 8800 9900 12100 13700

3500 5600 8600 10300 12200 14400 16100

1.15 1.23 1.15 1.17 1.16 1.21 1.15

a Conversion determined from the 1H NMR spectra of the reaction solution. bOverall degree of polymerization (DP) including both NiPAm blocks calculated from conversion and [M]/[CTA]. cOverall DP including both PNiPAm blocks calculated from suitable peak integrals in the 1H NMR spectra of the purified polymers. dSEC: chloroform/NEt3/isopropanol 94:4:2, RI detection, PS calibration.

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therefore a decrease of the transmittance of the mixtures. Surprisingly, this phase separation occurs at temperatures below 30 °C at a concentration of 5 g L−1. This is unexpected, since a PNiPAm homopolymer would undergo its coil to globule transition at a Tcp of 32 °C.9,10 Although representing a thermoresponsive polymer as well, the PEtOx block would still be well solvated in this temperature range. PEtOx homopolymers exhibit LCST behavior above 65 °C with a strong influence of the molar mass.17 A PEtOx with a DP of 20 even remains soluble until a temperature of 100 °C in pure water. Hence, we had expected the Tcp of all polymer solutions to be above 32 °C, while polymer solutions with shorter PNiPAm blocks would demix at even higher temperatures. However, the reverse trend was found: The Tcp increases with increasing DP of the NiPAm block. This trend is more pronounced for the shorter block copolymers, while solutions of polymers with longer PNiPAm blocks approach the Tcp of a PNiPAm solution. As expected, the Tcp of all polymer solutions increases with decreasing polymer concentration and the transition becomes less sharp. However, the trend within the series P1 to P6 remains the same. The phase separation of all polymer solutions is reversible during three heating/cooling cycles, as shown by the overlay of heating and cooling curves for selected samples in Figure 7. The respective data for all measurements are provided in the Supporting Information (Figures SI 5 to SI 7). A small hysteresis of 2−4 °C between the heating and cooling curves is observed for all polymer solutions. This is a common observation and a result of the fact that turbidimetry represents a dynamic method due to the heating rate applied during the measurements. In particular for PNiPAm-based thermoresponsive polymers, the formation of mesoglobules plays a significant role during this process.9 The hysteresis is more

Figure 5. 1H NMR spectra of the purified triblock copolymer P1 in D2O (red) and CDCl3 (black) and structural assignments of the peaks.

this field of research.9,10,42 Turbidimetry measurements were performed to investigate the thermoresponsive behavior of the triblock copolymers in aqueous media with a heating and cooling rate of 1 K min−1. Three heating/cooling cycles were conducted in a temperature range between 1 and 100 °C. The obtained transmittance curves from the second heating run are depicted in Figure 6. As common for the LCST behavior, all polymers are completely dissolved at low temperatures, while an increase of temperature results in phase separation and

Figure 6. Transmittance curves of solutions of P1 to P6 at different concentrations in water (second heating run, heating rate 1 K min−1; see Figure SI 4 for the full temperature range).

Figure 7. Heating/cooling hysteresis during selected turbidimetry measurements in water (second heating/cooling run, heating/cooling rate 1 K min−1). F

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Macromolecules Table 2. Cloud Point Temperatures Tcp of Various Aqueous Solutions of the Block Copolymers P1 to P6a Tcp (°C) H2O c (g/L) P1 P2 P3 P4 P5 P6

DP NiPAm 16 32 54 64 84 98

5 13.3 21.5 26.2 28.4 29.4 30.2

c

D2O

0.5 M NaClO4 b

1

0.5

5

5

20.2 26.7 29.9 31.5 32.3 32.8

25.5 29.8 32.9 34.8 35.3 35.7

14.9 23.9 27.0 30.4 31.7 32.2

27.6 22.4 24.0 24.9 24.9 25.0

0.5 M Li2SO4 b

1

5

31.2 29.0 29.0 29.0 29.6 29.0

1.0 2.7 3.5 5.3 5.5 5.6

1b 4.4 4.4 8.2 8.7 10.8 10.5

Heating rate 1 K min−1, Tcp reported at 50% transmittance. Values are extracted from the second heating run, unless noted otherwise. Tcp decreasing with the number of heating/cooling cycles. cValue reported for the first heating run due to the formation of aggregated structures during the first cycle (see also Figure SI 9). Second heating run: Tcp = 28.2 °C; third heating run: Tcp = 27.3 °C. a b

solvated as a result of hydrogen bonds between the polymer chains and water. As soon as phase separation occurs at higher temperatures, structures with increased hydrodynamic diameters were found, which underwent shrinkage upon further temperature increase. This is common for many polymer/water mixtures with LCST behavior and is caused by the different compositions of the two phases that are formed during demixing.19,43 The original size distributions are obtained again after cooling for P3 to P6, albeit at lower temperatures in some cases. This is not the case for P2, where the concentrated phase droplets only gradually decrease their size during cooling, so that structures with hydrodynamic diameters of around 130 nm remain present even at 10 °C. Remarkably, indications for phase separation in the aqueous solutions of P2 to P6 were found by DLS at temperatures well below the Tcp from turbidimetry. Cryo-TEM investigations were performed to obtain a deeper understanding of the aggregation in solution from both the clear solutions below Tcp and the turbid mixtures above Tcp. Samples were preheated in the climate controlled preparation chamber of the vitrification device (Vitrobot). Figure 9 depicts

pronounced at higher polymer concentrations. Although the phase transition for the triblock copolymer P1 with the shortest PNiPAm blocks is also reversible, the remixing during the cooling process is significantly slower compared to all other polymer solutions (compare Figure SI 8). The cloud point temperatures (Tcp) of the three different concentrations of the triblock copolymers P1 to P6 in deionized water are summarized in Table 2. DLS measurements were performed at different temperatures below and above the Tcp for all triblock copolymers P1 to P6 at a concentration of 1 g L−1 to elucidate the origin of the unexpected LCST behavior. In contrast to turbidimetry, no dynamic method was applied here as an equilibration time of 6 min was applied at each temperature prior to the measurement. It should also be noted that the samples were stirred during the turbidimetry measurements, while this was not the case during the DLS studies. Both heating and cooling of the solutions were investigated. The obtained volume size distributions are depicted for P1 to P3 in Figure SI 9. P1 forms aggregated structures with a hydrodynamic diameter of 150 nm already far below the Tcp of the solution, hinting toward the formation of loose aggregates18 of several polymer chains instead of complete solvatization of the isolated macromolecules. Above the Tcp of the solution, larger aggregates with a stable size of 750 nm are formed, which do not shrink upon further temperature increase (Figure 8). During cooling,

Figure 9. Cryo TEM images of the aqueous solution of P4 below (after cooling) and above Tcp (28 °C).

the obtained images acquired for P4 vitrified at different temperatures of the solution. In accordance with the DLS results, only very few loose aggregates could be barely found as indicated by the arrow heads (Figure 9, left). On the other hand, a sample vitrified at 50 °C clearly shows concentrated phase droplets in a matrix of the phase with lower polymer concentration.19 They feature a round structure with only a weak contrast and no sharp borders, indicating the formation of globuli. Even though a triblock copolymer is used no evidence for the formation of phase separated, ordered (hierarchical) structures (like micelles and vesicles or core-shell-corona structures) are observed. The size of the aggregates is rather disperse but is

Figure 8. Hydrodynamic diameters found at varying temperatures in the aqueous solutions of the triblock copolymers P1 to P6 at a concentration of 1 g L−1 as observed by DLS (volume size distribution, average value of three measurements). Left: P1. Right: P2 to P6.

aggregates with the original sizes are re-formed, which is in agreement with the results obtained from turbidimetry (i.e., the large heating/cooling hysteresis and the difference of the Tcp in the first and second heating run). At low temperature, DLS measurements revealed hydrodynamic diameters below 10 nm for P2 to P6 because the polymer chains are completely G

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decreases, which is in accordance with the observations made by DLS (i.e., the presence of aggregated structures already below the Tcp as observed by turbidimetry). Upon further elevation of the temperature especially the intensity of signal A assigned to the PNiPAm block decreases rapidly. The same holds true for peak C, which also indicates the collapse of the PNiPAm blocks. Interestingly, signal B, which is derived from the PEtOx middle block, cannot be detected above the Tcp of P6, thereby confirming the assumption that this block is not solvated above Tcp anymore. Upon further temperature elevation until 70 °C, the signals assigned to the PEtOx block do not reappear. However, peaks attributed to the PEtOx block remain visible in the spectra above the Tcp of P1, the triblock copolymer with PNiPAm blocks shorter than the central PEtOx block (Figure SI 11). The presence of hydrogen bonds between the two distinct block types would therefore shield the PEtOx block from an interaction with water molecules if the DP of the PNiPAm blocks is large enough. The aggregates formed between both block types could then act as hydrophobic segments that have to be kept in solution solely by the PNiPAm blocks not involved in hydrogen binding with the central PEtOx block. This could explain the lowered Tcp compared to PNiPAm homopolymers, the trend of the Tcp values within the triblock copolymer series P1 to P6, and the presence of aggregated structures below Tcp as observed by DLS. Based on this hypothesis, further turbidimetry experiments were conducted in the presence of salts. Anions as well as cations can influence the Tcp of solutions of thermoresponsive polymers due to their capability to enhance hydrophobic interactions according to the Hofmeister series.48 The underlying effects are well investigated, in particular for PNiPAm,49−51 but known to occur in POx solutions as well.52,53 For this purpose, two different polymer concentrations and two salts, i.e., lithium sulfate and sodium perchlorate, were examined (Table 2 and Figure 11). Lithium sulfate as a kosmotropic salt destabilizes the hydrogen bonds between the polymer and the water molecules for both homopolymer types present in the triblock copolymers P1 to P6. Because of the enhanced hydrophobic interactions

in agreement with the values obtaines by DLS measurements (150−200 nm). Also, the presence of aggregated structures in the aqueous solution of P1 below Tcp could be confirmed by cryo-TEM measurements (Figure SI-10). To gain structural information about the reason for the observed effects on the molecular level, 1H NMR measurements were performed in D2O (Figure 5 and Figure SI 3). Although all signals of the triblock copolymer could be detected, in particular the signals assigned to the PEtOx block are less intense compared with the 1H NMR spectrum measured in CDCl3. This hints toward the fact that the PEtOx block is not well solvated in D2O, which is surprising because PEtOx is more hydrophilic than PNiPAm. This observation holds true for polymers with short as well as long PNiPAm blocks, i.e., P1 as well as P6. The formation of hydrogen bonds between the two blocks could explain this phenomenon since PNiPAm displays a hydrogen bond donating moiety due to its secondary amide functionality. These hydrogen bonds would occupy binding sites for water molecules on the PEtOx block and, thus, diminishing its solvatization leading to lowered Tcp values. A similar phenomenon is reported in random copolymers of NiPAm and methacrylic acid44 as well for several POx-based polymer architectures that contain additional hydrogen bond donating moieties.45−47 Hence, 1H NMR measurements were conducted at varying temperatures below and above the Tcp of the polymer solutions. To ensure a straightforward comparison between the results obtained from the different methods applied, turbidimetry experiments were conducted in D2O prior to the NMR studies. Indeed, the Tcp values are slightly elevated in D2O compared with water at the same concentration (Table 2). Figure 10 shows an overlay of the 1H NMR spectra of P6 measured in D2O subsequent to normalization according to the residual solvent peak. A similar plot for P1 is provided in the Supporting Information (Figure SI 11). Characteristic signals for both blocks, i.e., PNiPAm and PEtOx, are marked in Figure 10 to demonstrate the effect of temperature increase on each part of the triblock copolymer. Already at a temperature of 303 K (slightly below the Tcp of the solution at 305 K), the intensity of the signals in the 1H NMR spectrum slightly

Figure 10. Left: 1H NMR spectra of P6 in D2O at varying temperatures below and above Tcp (c = 5 g L−1). The signal of the solvent is normalized to 1. For clarity, only selected signals are assigned to the copolymer structure. A complete assignment of all peaks is provided in Figure 5. Right: values of the integrals obtained from 1H NMR spectra at different temperatures. A zoom into the aromatic region of the spectra is shown in Figure SI 13. H

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Figure 11. Influence of salt addition on the cloud point temperatures of aqueous solutions of P1 to P6 at different concentrations in different solutions. Left: c = 5 g L−1. Right: c = 1 g L−1.

caused thereby, the Tcp value is decreased for all polymer solutions in comparison with pure water. As a result, the supposed intra- or intermolecular hydrogen bonds between the amide moieties of PNiPAm and PEtOx would be strengthened (or not affected). Indeed, the Tcp trend of the within the series P1 to P6 remained unaffected. The addition of sodium perchlorate is more interesting since this salt was reported to cause a slight salting-out effect for PNiPAm (resulting in a decrease of the Tcp)51 and a rather strong salting-in effect for POx (elevated Tcp)52 homopolymers at a salt concentration of 0.5 mol L−1. Accordingly, a significant increase of the Tcp of the solution of P1 was observed, as P1 represents the only polymer with a higher mole fraction of PEtOx than PNiPAm (NiPAm8−EtOx20−NiPAm8). Hence, P1 would expose PEtOx moieties to the water molecules regardless of the presence of hydrogen bonds between both block types. This finding is in agreement with the results obtained from all other techniques discussed so far. Remarkably, also the Tcp of the solution of P2 (NiPAm16−EtOx20−NiPAm16) was slightly higher in aqueous sodium perchlorate solution than in pure water, hinting toward the fact that PEtOx moieties might still be accessible for surrounding water molecules and perchlorate anions. As an alternative, one might assume that ions can penetrate into the segments formed by hydrogen bonding between the PEtOx and the PNiPAm blocks.50 However, the Tcp remains constant for P3 to P6 in 0.5 M sodium perchlorate solution for both investigated polymer concentrations (at 25 °C at a concentration of 5 g L−1 and 29 °C at 1 g L−1), irrespective of the copolymer composition. Thus, the influence of the PNiPAm blocks becomes predominant in these cases.

on the thermoresponsive properties of the triblock copolymers (Figure 12). The secondary amide moieties of the PNiPAm

Figure 12. Schematic representation of the hydrogen bonds formed between the PEtOx and PNiPAm segments.

blocks are believed to act as donors to form hydrogen bonds with accepting carbonyl functionalities of the central PEtOx block, which prohibits a solvatization of the latter. Hence, triblock copolymers with short PNiPAm blocks form aggregated structures already below the Tcp of their aqueous solutions. The LCST behavior of the polymer solutions with longer PNiPAm chains is predominated by the PNiPAm blocks, which keep the partly collapsed macromolecules in solution below the Tcp. Future research will focus on the exploitation of the established synthesis method to prepare symmetrical triblock copolymers with other POx and POx of increased DP as well as other vinylic building blocks. The latter is particularly interesting to provide further insight into the suggested formation of hydrogen bonds between the two blocks types in aqueous solution, since thermoresponsive polymers without hydrogen bond donating moieties would presumably prevent a collapse of the macromolecules at low temperatures.

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CONCLUSION A series of well-defined symmetrical PNiPAm-b-PEtOx-bPNiPAm triblock copolymers was synthesized by RAFT polymerization using a bifunctional macro chain transfer agent. For this purpose, a benzyl bromide-based dual initiator was employed for the CROP of EtOx. Quantitative in situ functionalization of both living PEtOx chain ends with a carboxylic acid functional trithiocarbonate was confirmed via 1H NMR spectroscopy as well as ESI- and MALDI-ToF-MS. The LCST behavior of the final triblock copolymers in aqueous solutions was thoroughly investigated using turbidimetry, 1H NMR, DLS, and cryo-TEM measurements. All applied techniques consistently hint at the presence of hydrogen bonds between the PEtOx and PNiPAm segments, which exhibit a strong influence

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01371. ESI-ToF-mass spectrum of MCTA, 1H NMR spectra of P6 in D2O and CDCl3, 1H NMR spectra of P1 in D2O at varying temperatures, transmittance curves of aqueous solutions of P1 to P6 at three concentrations, transmittance curves of the aqueous solution of P1 for all three heating/cooling cycles, SEC traces of the triblock copolymers measured directly from the reaction solution, DLS measurements of the triblock copolymers P1 to P3 for the first heating and cooling cycle (PDF) I

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AUTHOR INFORMATION

Corresponding Author

*(U.S.S.) Fax +49(0) 3641 9482 02; e-mail ulrich.schubert@ uni-jena.de. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Nicole Fritz for the ESI-Q-ToF-MS and Annett Urbanek for the MALDI-ToF-MS measurements. We also thank Dr. Peter Bellstedt and Gabriele Sentis (FSU NMR-Plattform) for NMR measurements. The cryo-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).

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