Self-Association of a Thermosensitive Poly(alkyl-2-oxazoline) Block

Article pubs.acs.org/Macromolecules

Self-Association of a Thermosensitive Poly(alkyl-2-oxazoline) Block Copolymer in Aqueous Solution Rintaro Takahashi,† Takahiro Sato,*,† Ken Terao,† Xing-Ping Qiu,‡ and Françoise M. Winnik‡ †

Department of Macromolecular Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043, Japan Faculty of Pharmacy, Department of Chemistry, University of Montreal, CP 6128 Succursale Centre Ville Montreal, Quebec, Canada H3C 3J7

‡

S Supporting Information *

ABSTRACT: We have investigated the heat-induced self-association in water of a block copolymer (PIPOZ-b-PEOZ) comprising two thermosensitive blocks: a poly(2-isopropyl-2-oxazoline) block (degree of polymerization 71) and a poly(2ethyl-2-oxazoline) block (degree of polymerization 38) using differential scanning calorimetry (DSC), static light scattering (SLS), dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS) together with visual observation on the macroscopic and microscopic scales. The dehydration temperatures of the PIPOZ and PEOZ blocks in water are 43 and 54 °C, respectively. When heated abruptly to 50 °C where PIPOZ-b-PEOZ is amphiphilic, the copolymer first forms star micelles that further aggregate to form large concentrated phase droplets, and finally the droplets coalesce into a bulk liquid phase, having a copolymer concentration as high as 0.8 g/cm3. When heated abruptly to 70 °C, where both blocks are hydrophobic, the copolymer solution also separates into two liquid phases, consisting of phase-separated polymer-rich micrometer or submicrometer size droplets dispersed in a polymer-poor liquid phase, but the droplets do not coalesce into a liquid bulk phase. We discuss the role of the more hydrophilic PEOZ block in the macroscopic phase separation behavior. No microphase separation takes place in the concentrated phase of the aqueous PIPOZ-b-PEOZ block copolymer solution.

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INTRODUCTION Recently, extensive studies have been performed on the temperature-induced micellization in water of block copolymers consisting of hydrophilic and thermosensitive blocks.1−5 Poly(N-isopropylacrylamide) is a typical thermosensitive block.6 It has been coupled with various hydrophilic blocks including poly(ethylene glycol),7,8 polypeptides,9 poly(Nalkylacrylamide),10 poly(N-vinylpyrrolidone),11 and poly(acrylic acid).12 These diblock copolymers are soluble in cold water but form core−corona micelles when the solution temperature exceeds the cloud point temperature TCP of the aqueous PNIPAM solution. For such block copolymers, the interaction parameter between the thermosensitive block and the solvent can be changed continuously by raising or lowering the temperature. This change in interaction parameter makes it possible to control, through temperature, the size and morphology of micelles formed by such block copolymers.2−5 With the advent of novel controlled polymerization techniques, it has become possible to prepare block copolymers consisting of two thermosensitive block, known as doubly thermosensitive block copolymers.13 The thermoresponsiveness of such copolymers is quite different from the cases cited above, since the interaction parameters between each block and the solvent can change as a function of solution temperature. Aoshima et al.14 synthesized doubly thermosensitive block © 2012 American Chemical Society

copolymers of vinyl ethers with different TCP. The resulting copolymers in water were shown to exhibit unique physical gelation characteristics. Hua et al.15 studied doubly thermosensitive block copolymers with pendant oxyethylene moieties and reported their turbidity behavior in aqueous solution. However, detailed studies on the micellization and selfassociation behavior of such block copolymers in water have not been reported as yet. The study reported here pertains to the temperature-directed self-assembly in water of doubly thermosensitive diblock copolymers consisting of a poly(2-isopropyl-2-oxazoline) (PIPOZ) block and a poly(2-ethyl-2-oxazoline) (PEOZ) block (see Scheme 1). Solutions of either homopolymer, PIPOZ or PEOZ, undergo phase separation upon heating beyond their TCP. Depending on the polymer molecular weight and concentration, TCP ranges from 36 to 80 °C16 and from ∼62 to 100 °C17,18 for solutions of PIPOZ and PEOZ, respectively. Gradient copolymers of 2-ethyl-2-oxazoline and 2isopropyl-2-oxazoline [P(EOZ/IPOZ)] have been prepared by Park and Kataoka.19 Turbidity measurements carried out with solutions of the gradient P(EOZ/IPOZ) copolymers in Received: May 14, 2012 Revised: July 8, 2012 Published: July 23, 2012 6111

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DMF) and a TSK-gel α-3000 (particle size 7 μm, exclusion limit 1 × 105 Da for polystyrene in DMF) (Tosoh Biosep) columns, a Dawn EOS multiangle laser light scattering detector λ = 690 nm (Wyatt Technology Co.), and an Optilab DSP interferometric refractometer λ = 690 nm (Wyatt Technology Co.) under the following conditions: injection volume, 100 μL; flow rate, 0.5 mL/min; eluent, DMF; temperature, 40 °C. The refractive index increment ∂n/∂c of the diblock copolymer was calculated by ∂n/∂c = w1(∂n/∂c)1 + w2(∂n/∂c)2 with w1 and w2 denoting the weight fraction of PIPOZ (∂n/∂c = 0.084 cm3/g) and PEOZ (∂n/∂c = 0.078 cm3/g) blocks, respectively.26 The weight-average molecular weight Mw and the ratio of the weight- to number-average molecular weights Mw/Mn were determined in the routine procedure. Synthesis of α-Propargyl-ω-hydroxylpoly(2-isopropyl-2-oxazoline). The polymerization was conducted in acetonitrile at 80 °C according to the general procedure.27 To prepare PIPOZ, a roundbottom flask equipped with a N2-filled condenser and a rubber stopper was charged with 2-isopropyl-2-oxazoline (4.5 mL, 40 mmol), acetonitrile (20 mL), and propargyl p-toluenesulfonate (100 μL, 0.60 mmol) via oxygen-free syringes at room temperature. The flask was immersed in a preheated oil bath. The polymerization mixture was stirred at 80 °C for 48 h. At the end of polymerization, water (5 mL) was added to quench the poly(2-isopropyl-2-oxazoline) oxazolinium living end groups. The termination reaction was conducted at 80 °C for 6 h. The polymer solution was diluted with water to 100 mL and dialyzed against water for 3 days with a membrane with MWCO of 3500 Da. The purified polymer was recovered by freeze-drying. Yield 3.4 g, 75%. 1H NMR (CDCl3, δ) ppm: 1.10 (br, −CH(CH3)2), 2.66 and 2.89 (br, −CH(CH3)2), 3.45 (br, −NCH2CH2−), 4.16 (s, −NCH2CCH). FT-IR, 2976, 2934, 2873, 1646, 1474, 1431, 1205, 1160, 1089, and 755 cm−1. Molecular characteristics of two PIPOZ samples synthesized are listed in Table 1. The sample of Mw = 1.14 × 104 was used only for differential scanning calorimetry. High-Sensitivity Differential Scanning Calorimetry (HS-DSC). HS-DSC measurements were performed on a VP-DSC microcalorimeter (MicroCal Inc.) at an external pressure of ca. 180 kPa. The cell volume is 0.520 cm3. The heating rate was 1.0 °C/min. Scans ranging from 10 to 80 °C were recorded. Data were analyzed using the Origin based software supplied by the manufacturer. The polymer concentration was 1.0 × 10−3 g/cm3. The temperature of the phase transition TM was taken at the maximum of the endotherm. The enthalpy of the transition ΔH was determined from the area of the endothermic peak. For each experiment, three consecutive scans were performed. Macroscopic and Microscopic Observations. Aqueous PIPOZb-PEOZ and PIPOZ solutions (concentration ∼0.18 g/cm3) were placed in stoppered glass test tubes. They were heated at given temperatures in a thermostated water bath, and their turbidity was observed visually. For direct observation of the phase separation on the microscopic scale, a polymer solution (6 wt %) in water was held between two microscope slides as follows.28 The solution (10 μL) was placed on a glass slide; a smaller glass coverslip was placed on top of the drop. The edge of the coverslip was sealed with an epoxy glue. The liquid spread over a circular area ca. 10 mm in diameter. The solution thickness, estimated from the solution volume and the area of the spread solution, was ca. 100 μm. The slides were mounted on the temperature control stage (THMS 600 with TMS94, Linkam) of a microscope (Axioskop 2, Carl Zeiss) preheated to 50 °C and kept still at this temperature for 2 h prior to observation.

Scheme 1. Chemical Structure of the Block Copolymer PIPOZ-b-PEOZ Studied in This Worka

a

x is the mole fraction of 2-isopropyl-2-oxazoline units, and N is the degree of polymerization of the copolymer.

phosphate buffer saline (PBS, pH 7.4) indicated that TCP varied linearly, from 38 to 67 °C, as the EOZ content of the copolymer increased from 0 to 75 mol %. The authors also carried out static light scattering (SLS) studies of the solutions with changing temperature. They found no evidence for the formation of micellar assemblies in these solutions, which indicates that the significant change of the chain amphiphilicity from one chain end to the other is not sufficient to trigger micelle-like association P(EOZ/IPOZ) gradient copolymers. For the diblock copolymer PIPOZ-b-PEOZ, one may anticipate that micellization will occur within the limited temperature range, for which water is a selective solvent, and that macroscopic phase separation will take place above this temperature range, where water becomes nonsolvent. The study reported here addresses this issue, which is of importance from the point of view of polymer theory, but also from the practical viewpoint, given the possible uses of such copolymers.20−25 The self-association process was monitored on the macroscopic scale, by visual observation of PIPOZ-b-PEOZ solutions heated and cooled between 20 and 70 °C and by observation of solutions placed in the temperature-controlled stage of an optical microscope, by differential scanning calorimetry (DSC), and on the nanoscale, via SLS and smallangle X-ray scattering (SAXS). Particular attention was devoted to assessing, in aqueous PIPOZ-b-PEOZ solutions, the competition between micellization and macroscopic phase separation brought about by changes in temperature.

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EXPERIMENTAL SECTION

Materials. Isobutyric acid (99.0%), 2-aminoethanol (99.0%), 2ethyl-2-oxazoline (99%), calcium hydride, propargyl p-toluenesulfonate (97.0%), and sodium sulfate (Na2SO4, anhydrous) were purchased from Sigma-Aldrich Chemical Co. 2-Isopropyl-2-oxazoline was synthesized from isobutyric acid and 2-aminoethanol according to a previous report.24 2-Ethyl-2-oxazoline was dried over calcium hydride and distilled under vacuum. Acetonitrile was purified by distillation over calcium hydride. All other solvents were of reagent grade and used as received. Water was deionized using a Millipore Milli-Q system. Instrumentation for Sample Characterization. 1H NMR spectra were recorded on a Bruker AMX-400 (400 MHz) spectrometer using chloroform-D as the solvent. Gel permeation chromatography (GPC) was performed on a GPC-MALLS system consisting of an Agilent 1100 isocratic pump, a set of TSK-gel α-M (particle size 13 μm, exclusion limit 1 × 107 Da for polystyrene in

Table 1. Molecular Characteristics of PIPOZ and PIPOZ-b-PEOZ Samples Synthesized polymer PIPOZ PIPOZ-b-PEOZ

Mw/103 a

Mw/Mna

7.1 11.4 11.8

1.04 1.08 1.05

xIPOZb

Nwc

TM/°C

ΔHd

0.65

63 101 71e/38f

46.7 43.2 43.2e/54.4f

4.3 5.4 1.6e/1.5f

a

Measured by GPC-MALLS. bMole fraction of IPOZ in the copolymer measured by 1H NMR. cWeight-average degree of polymerization. dIn units of kJ/mol of monomer. eFor the PIPOZ block. fFor the PEOZ block. 6112

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Light Scattering. Aqueous solutions of PIPOZ-b-PEOZ and PIPOZ of concentration ∼0.1 10−3 g/cm3 were transparent, so light scattering measurements could be performed without undesirable effects usually associated with sample turbidity. Static light scattering (SLS) and dynamic light scattering (DLS) measurements were done using a ALV/SLS/DLS-5000 light scattering goniometer with vertically polarized light of 532 nm wavelength. The light scattering system was calibrated using toluene as the reference material. Test solutions of PIPOZ-b-PEOZ and PIPOZ were filtered at room temperature into cylindrical light scattering cells (with a ca. 14 mm diameter) using a membrane filter with a pore size of 0.2 μm. The filled cells were immersed into the xylene bath of the LS sample compartment preheated to 50 or 70 °C. The temperature of the solution within the cells reached 50 or 70 °C ca. 5 min after immersion of the cell into the bath, at which point scattering intensity measurements were initiated. Scattering intensities were determined as a function of the time elapsed after the temperature jump (T-jump) at three scattering angles: θ = 30°, 40°, and 50°. In order to check the reproducibility of the measurement, T-Jump experiments to a given temperature were repeated several times using solutions which had not been heated previously. The SLS data were analyzed to determine the weight-average molar mass Mw, z-average particle scattering factor Pz(k), and z-average radius of gyration ⟨S2⟩z1/2, using the Guinier plot ln(R θ /Kc) = ln[M w Pz(k)] = ln M w −

1 2 2 ⟨S ⟩z k + O(k 4) 3

spectral data, by comparing the areas of the resonance due to the isopropyl methine proton (2.66−2.89 ppm) to that of the ethyl methylene protons (2.32−2.42 ppm) (Figure S3). The polymerization degree of the PIPOZ block (62) was estimated from the ratio of the areas of signals due to the resonance of the isopropyl methine protons to that of the propargyl methylene protons (4.16 ppm). This value is nearly identical to the monomer/initiator feed ratio (60) and to Nw determined from the GPC-MALLS traces. Aqueous solutions of both PIPOZ and PIPOZ-b-PEOZ were clear at room temperature. They became turbid when heated past a temperature of ∼42 °C. The cloud points of PIPOZ and the PIPOZ-b-PEOZ sample in water (1.0 × 10−3 g/cm3) were 44.6 and 44.0 °C, as determined from the temperature-driven changes in the solution transmittance at 550 nm (Figure S4). Phase Transition of PIPOZ-b-PEOZ in Water Observed by Microcalorimetry. A premise of this study is that there exists a temperature for which water is a nonsolvent for the PIPOZ block and a solvent for the PEOZ block or, in other words, that the dehydration of the two blocks occurs at two different temperatures. Thermograms recorded by HS-DSC for aqueous solutions (1.0 × 10−3 g/cm3) of PIPOZ-b-PEOZ, PIPOZ (Mw = 11 400) and PIPOZ (Mw = 7000) are presented in Figure 1. The thermogram corresponding to the diblock

(1)

where Rθ is the excess Rayleigh ratio of the solution, K is the optical constant, c is the polymer mass concentration, and k2 is the square of the scattering vector. Since the polymer concentration examined was low (c = 1.0 × 10−4 g/cm3), the intermolecular interference effect was neglected in the above equation. Specific refractive index increments ∂n/∂c were measured using a differential refractometer of the modified Schulz−Cantow type. The values were 0.172 and 0.170 cm3/g for PIPOZ-b-PEOZ in water at 25 and 40 °C and 0.160 cm3/g for PIPOZ in water at 50 °C. The results for PIPOZ-b-PEOZ were extrapolated to higher temperatures to obtain the increments at 50, 56, and 70 °C. The optical constants K were calculated by using these refractive index increments. Small-Angle X-ray Scattering (SAXS). SAXS measurements were conducted on PIPOZ-b-PEOZ aqueous solutions of concentration c = 0.026 g/cm3 kept at 50 and 70 °C and on a solution of PIPOZ of concentration c = 0.0244 g/cm3 at 50 °C, using the beamline 40B2 of SPring-8, Kobe, Japan. Solutions were poured in a capillary at room temperature and rapidly heated to the desired temperature by setting the capillary in the heating block of the sample holder. The intensity of the scattered X-ray was measured as a function of the time elapsed after the T-jump using an imaging plate detector.

Figure 1. DSC thermograms of aqueous solutions of PIPOZ-b-PEOZ (open circles) and of PIPOZ with Nw = 101 and 63 (full circles); c = 1.0 × 10−3 g/cm3. For the sake of clarity, the scales of the two ordinates (molar heat capacity) are not the same; the left-hand side axis corresponds to the PIPOz-b-PEOZ thermogram, while the PIPOZ thermograms are linked to the right-hand side axis, as indicated by arrows on the curves. The dotted curves are simulated curves of each component (PIPOZ and PEOZ) of the experimental trace of the diblock copolymer (open circles) obtained using two Cauchy− Gaussian functions.

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RESULTS AND DISCUSSION Synthesis of the Polymers. The diblock copolymer PIPOZ-b-PEOZ was obtained by sequential cationic ringopening polymerization (CROP) of 2-isopropyl-2-oxazoline and 2-ethyl-2-oxazoline initiated with propargyl tosylate (Figure S1).22 The living polymerization characteristics of 2-alkyl-2oxazolines CROP with this initiator have been reported previously.22 Kinetic plots obtained in our laboratory confirmed that in our hands the polymerization proceeded similarly. The molecular weights of PIPOZ and the corresponding diblock copolymer PIPOZ-b-PEOZ (Table 1) were determined by GPC-MALLS. The GPC elution band corresponding to PIPOZ-b-PEOZ was shifted to a shorter elution time, compared to that of the PIPOZ precursor (Figure S2). It presented a small shoulder at the elution time of the PIPOZ precursor, indicating the presence of a small amount of residual PIPOZ. The molar fraction (x) of 2-isopropyl-2-oxazoline in the PIPOZ-b-PEOZ sample was evaluated from 1H NMR

copolymer presents two distinct endotherms with maxima at TM = 43.2 °C (endotherm 1) and 54.4 °C (endotherm 2). The two endotherms are well separated, with a minimum in heat capacity at ∼50 °C. Each endotherm is broad and markedly asymmetric, with a sharp increase of the heat capacity on the low-temperature side and a gradual decrease of the heat capacity for temperatures higher than TM. Endotherm 1 is attributed to the dehydration of the PIPOZ block. The maximum of this endotherm coincides with that of PIPOZ (11K), a polymer of weight-average degree of polymerization Nw = 101, a value close to the total Nw (109) 6113

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Figure 2. Visual appearance of aqueous solutions of (A) PIPOZ-b-PEOZ (c = 0.18 g/cm3) at 50 °C, (B) PIPOZ-b-PEOZ (c = 0.19 g/cm3) at 70 °C, and (C) PIPOZ (7K) (c = 0.18 g/cm3) at 50 °C, and microscopic images of aqueous solutions of (D) PIPOZ-b-PEOZ (c = 0.059 g/cm3) at 70 °C, and (E) PIPOZ (7K) (c = 0.059 g/cm3) at 50 °C; the samples were kept at the given temperatures for 240 min in panels A−C and for 120 min in panels D and E.

study of (poly(ethylene glycol))-grafted poly(N-isopropylacrylamides). They attributed this observation to the fact that a fraction of the NIPAM units do not dehydrate as a consequence of the presence of PEG chains. The hydrated PEOZ block may affect similarly the transition of the PIPOZ block. Further experiments are in progress to understand the origin of the effect. Phase Separation of PIPOZ-b-PEOZ on the Macroscopic and Microscopic Scales. Aqueous solutions of PIPOZ-b-PEOZ and PIPOZ (c ≈ 0.18 g/cm3) were heated either to 50 °C, a temperature above the phase transition of the PIPOZ block or to 70 °C, a temperature where water is a nonsolvent for both blocks. The solutions became turbid upon heating past ∼45 °C. Upon prolonged heating (240 min) at 50 °C, the PIPOZ-b-PEOZ solution underwent macroscopic liquid−liquid phase separation (Figure 2, panel A). The two liquid phases were separated and placed in two different vials. The copolymer concentration of the nearly transparent lower liquid phase was 0.79 g/cm3, as determined by densitometry. Since the upper liquid phase was slightly turbid, we did not determine its concentration. The turbidity of this phase may arise from the presence of micelles (see below) that did not sediment by gravity. Panel C of Figure 2 shows a vial containing the homopolymer (PIPOZ) solution kept at 50 °C for 240 min. The sample was turbid, with no sign of macroscopic liquid/ liquid phase separation. Similarly (Figure 2, panel B), the PIPOZ-b-PEOZ solution remained turbid with no sign of liquid/liquid phase separation after being kept at 70 °C for the same period of time. (Figure 2, panel B). Next, we monitored the phase separation by optical microscopy. Sealed samples of a polymer solution were placed in the heating stage of a microscope and brought to 50 or 70

of PIPOZ-b-PEOZ (see Table 1). It is shifted to lower temperature, compared to the endotherm of PIPOZ (7K), the homopolymer of Nw (63), close to Nw (71) of the PIPOZ block of the copolymer (Figure 1). The phase transition of PIPOZ is known to show a marked inverse dependence on molecular weight.16 The temperature of the phase transition of the PIPOZ block reflects the molecular weight of the copolymer in its entirety, rather than that of the PIPOZ block. Endotherm 2 of the PIPOZ-b-PEOZ solution, centered at 54.4 °C, is assigned to the dehydration of the PEOZ block. This transition takes place at a temperature for which the PIPOZ block is insoluble; hence the PEOZ blocks, linked to aggregated PIPOZ segments, will experience a high local concentration, a situation similar to that of polymer chains in the corona of core−shell micelles. Indeed, in their study of polyion complex micelles with a PIPOZ corona, Park et al.29 observed that the phase transition of the PIPOZ block occurs at a temperature significantly lower than that of the corresponding PIPOZ homopolymer. A similar depression of TM is observed here for endotherm 2: the TM for PEOZ of Nw ∼ 100 is known to exceed 90 °C.19 Thermodynamic data obtained from the thermograms of PIPOZ and PIPOZ-b-PEOZ are listed in Table 1. To evaluate the transition enthalpy corresponding to each endotherm, the copolymer thermogram was fitted to two Cauchy−Gaussian curves, represented by dotted lines in Figure 1. Surprisingly, the transition enthalpy ΔH (per monomer unit) for the PIPOZ block was calculated to be ∼1/3 of the enthalpy of the PIPOZ (homopolymer) transition. Although it is known that the ΔH value is affected by PIPOZ chain length (see top entries Table 1), the magnitude of the decrease in enthalpy is too large to reflect only molecular weight effects. A substantial decrease in the transition enthalpy was reported by Virtanen et al.30 in their 6114

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Figure 3. Time evolution of Mw and ⟨S2⟩z1/2 for aggregates formed in aqueous PIPOZ-b-PEOZ (panels A−C) and PIPOZ (panel D) solutions (c = 1.0 × 10−4 g/cm3) after T-jump to a set temperature indicated in each panel. For each temperature, three independent T-jump experiments were performed. In each panel three different symbols are used to indicate results obtained independently under a given set of experimental conditions.

were heated rapidly from room temperature to 50, 56, or 70 °C (T-jump experiment). Subsequently, changes of the weightaverage molar mass, Mw, and the z-average radius of gyration, ⟨S2⟩z1/2, were monitored as a function of time using eq 1 and Rθ values at θ = 30°, 40°, and 50° (Figure 3A−C). In each panel of Figure 3, three different symbols are used to indicate results obtained from three independent measurements carried out under identical experimental conditions. Focusing first on Mw data recorded upon subjecting PIPOZ-b-PEOZ solutions to T-jumps, we note that, within a given set of conditions, results show very poor reproducibility. Trends in radii of gyration data are more consistent: in all cases ⟨S2⟩z1/2 increased over the first 15 min post T-jump, reaching a plateau value of 100−200 nm. Although the ⟨S2⟩z1/2 data were more reproducible than Mw values, the differences from run to run were still rather large. Control SLS experiments were performed with aqueous solutions of the homopolymer (PIPOZ). Experimental results recorded upon subjecting PIPOZ solutions to T-jumps from room temperature to 50

°C. When the PIPOZ-b-PEOZ solution was kept at 70 °C for 120 min, droplets were detected (Figure 2D), although on the macroscopic scale the copolymer solution did not form two well-separated phases. The droplet size ranged from ∼10 to >100 μm. Droplets were also observed by microscopy when a sample of aqueous PIPOZ was kept at 50 °C for 120 min (Figure 2E). In this sample, the droplet size distribution was bimodal, with a population of large droplets (∼20−25 μm in diameter) and a population of small droplets (diameter