Note pubs.acs.org/Macromolecules
Changes in Nanoassembly of Oligocaprolactone End-Capped Pluronic F127 and the Abnormal Hydrophobicity Trend of Phase Transition Yu Kyung Jung, Min Hee Park, Hyo Jung Moon, Usha Pramod Shinde, and Byeongmoon Jeong* Department of Chemistry and Nano Science, Department of Bioinspired Science (WCU), Global Top 5 Program, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea S Supporting Information *
ABSTRACT: Compared with unmodified F127, the concentration range exhibiting sol−gel transition increased for the CL4-F127-CL4 (F-CL4); however, it decreased for the CL12-F127-CL12 (F-CL12), even though both F-CL4 and F-CL12 were hydrophobically modified by the oligocaprolactone (OCL). To understand the abnormal behavior of the OCL end-capped F127, the difference in basic nanoassemblies among the F127, F-CL4, and F-CL12 were investigated at a low concentration of 0.10 wt % as well as at high concentrations exhibiting sol−gel transitions. Dynamic mechanical analysis, 13 C NMR spectroscopy, hydrophobic dye solubilization, dynamic light scattering, microcalorimetry, and transmission electron microscopy suggested that F127’s undergo a unimer-to-micelle transition, whereas F-CL4’s undergo unimer-to-vesicle transition as the temperature increased. On the other hand, F-CL12’s with relatively large end-capped OCL formed aggregated micelle structures that undergo temperature-sensitive conformation changes. In addition, as the temperature increased, sol-to-gel-tosyneresis and gel-to-sol-to-gel-to-syneresis transitions were observed for F-CL4 and F-CL12 aqueous solutions, respectively, whereas a sol-to-gel-to-sol transition was observed for Pluronic F127 aqueous solution. The findings suggest that the end-capping of F127 by OCL induces changes in nanoassemblies, which play a key role in different physicochemical properties leading to the abnormal phase behavior.
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INTRODUCTION Thermogelling polymer aqueous solutions have been extensively investigated for their unique sol−gel transition mechanisms and also for their potential biomedical applications including drug delivery and tissue engineering.1−5 One of the classical examples is a poly(ethylene glycol)−poly(propylene glycol)−poly(ethylene glycol) triblock copolymer (Pluronic) aqueous solution. Among the Pluronics, (ethylene glycol)99− (propylene glycol)67−poly(ethylene glycol)99 (F127) exhibits an excellent thermogelling property that is attributed to a hydrophilic−hydrophobic balanced structure with a proper molecular weight of each block.6 The modification of Pluronic has been actively investigated as a means of varying physicochemical properties of the polymer including gel duration, sol−gel transition temperature, gel window, and critical gel concentration.7−16 In particular, when the hydroxyl end groups of Pluronic were modified by biodegradable poly(lactic acid) or polycaprolactone, the concentration at which sol-to-gel transition occurred increased even though the hydrophobicity of the polymer increased.7,9,10 The underlying mechanism was suggested to be based on the interference of the sol-to-gel transition mechanism of the original unmodified Pluronic. Unmodified Pluronics undergo a unimer-to-micelle transition in water as the temperature © 2013 American Chemical Society
increases; when the micelle fraction is greater than 0.53, the polymer aqueous solution turns into a gel.17,18 In the gel state, micelles of F127 are packed in a cubic lattice.17,18 The endcapped polymers of the Pluronic were suggested to interfere the micelle packing mechanism and thus hampered the sol-to-gel transition, resulting in the increase in the concentration at which the sol-to-gel transition occurred.7,9,10 However, the proposal is partially true and needed to be elaborated up to a certain range of the molecular weight of end-capped polymer. To understand the details of the interferences, we hypothesized that there might be differences in self-assembly at low concentrations, depending on the molecular weight of hydrophobically end-capped polymers. In this research, we synthesized a series of oligocaprolactone (OCL) end-capped Pluronic F127 (F-CLs) with a various number of caprolactone repeating units from 4 to 23. The physicochemical properties of the neat polymers and polymer assemblies in water were investigated by X-ray diffractometry, differential scanning calorimetry, dynamic mechanical analysis, scanning electron microscopy (SEM), nuclear magnetic resonance (NMR) Received: February 5, 2013 Revised: April 23, 2013 Published: May 6, 2013 4215
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Table 1. List of Polymers Studied code
polymera
Mna
Mnb
PDIb
Tmc (°C) peak/range
ΔHmc (J/g)
F127 F-CL4 F-CL8 F-CL10 F-CL12 F-CL15 F-CL23
CL0-F127-CL0 CL4-F127-CL4 CL8-F127-CL8 CL10-F127-CL10 CL12-F127-CL12 CL15-F127-CL15 CL23-F127-CL23
12 600 13 510 14 420 14 880 15 340 16 020 17 840
10 870 11 370 13 100 13 330 13 520 13 790 14 810
1.3 1.2 1.2 1.2 1.3 1.2 1.3
54.4/50.3−56.3 48.4/45.7−50.3 46.2, 47.8/43.5−49.0 45.6, 47.2/43.3−48.3 46.0, 47.2/43.4−48.3 44.4, 46.7/42.1−48.0 43.7, 46.1/41.3−47.4
105.9 74.8 79.0 73.6 55.7 61.4 56.6
a Determined by 1H NMR in CDCl3. Number-average molecular weight (Mn) of F127 is 12 600 Da. bDetermined by gel permeation chromatography. N,N-Dimethylformamide was used as an eluting solvent, and PEGs in a range of 400−20 000 were used as molecular weight standards. Polydispersity index (PDI) was defined by the Mw/Mn. cDetermined by differential scanning calorimetry. Heating rate was 3.0 °C/min. The melting point (Tm) and the enthalpy of melting (ΔHm) were analyzed by the second heating curve of the thermogram of the each polymer.
aqueous polymer solution (1.0 mL) was put in a vial with an inner diameter of 11 mm. The transition temperatures were determined by a flow (sol)−no flow (gel) criterion when the test tube was inverted with a temperature increment of 1 °C per step. The gel-to-syneresis transition temperatures were determined by visual observation of phase separation between polymer and water. Each data point is an average of three measurements, and the transition temperatures were reproducible within ±1 °C. Dynamic Mechanical Analysis. Changes in modulus of the polymer aqueous systems were investigated by dynamic rheometry (Thermo Haake, Rheometer RS 1). The aqueous polymer solution in a sol state was placed between parallel plates of 25 mm diameter with a gap of 0.5 mm and kept at 4 °C for 1 h. The data were collected under conditions of controlled stress (4.0 dyn/cm2) and a frequency of 1.0 rad/s. The heating rate was 0.5 °C/min. To minimize water evaporation during the experiment, the plates were enclosed in a water saturated chamber. Scanning Electron Microscopy. To investigate the gel morphologies, the polymer solutions (F127: 24.0 wt %; F-CL4: 24.0 wt %; and F-CL12: 9.0 wt %) were dropped on the silicon wafer in a vial, and the closed vial was kept at 37 °C in the oven for 10 min. Then, they were quenched into liquid nitrogen at −196 °C and then freeze-dried. SEM images were obtained using a JSM-6700F field emission SEM (JEOL). UV−vis Spectroscopy. 1,6-Diphenyl-1,3,5-hexatriene solution in methanol (10 μL at 0.4 mM) was injected into a polymer aqueous solution (1.0 mL at 0.10 wt %) to make the dye concentration of 4.0 μM. The UV−vis spectra (S-3100, SCINCO) were compared as a function of temperature in a range of 5−60 °C.12,18 Dynamic Light Scattering. The apparent sizes of the polymer and polymer assemblies in water (0.10 wt %) were studied by a dynamic light scattering instrument (ALV 5000-60x0) as a function of temperature in a range of 5−60 °C. A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm was used as a light source. Measurements of the scattered light were made at an angle of 90° to the incident beam. The results of dynamic light scattering were analyzed by the regularized CONTIN method. The decay rate distributions were transformed to an apparent diffusion coefficient. From the diffusion coefficient, the apparent hydrodynamic size of a polymer aggregate can be obtained by the Stokes−Einstein equation. Microcalorimetry. A microcalorimeter (Microcal, VP-DSC) was used to study heat exchange of the polymer aqueous solution (0.10 wt %) in a temperature range of 5−60 °C with a heating and cooling rate of 1.0 °C/min. Distilled water (0.5 mL) at 5 °C was loaded in a reference cell, and the polymer aqueous solution (0.4 mL) at 5 °C was loaded in a sample cell. The heating cycle started from 5 °C (T1) to 60 °C (T2) in the prestabilized instrument at 5 °C, and the thermogram was recorded. Transmission Electron Microscopy. A polymer aqueous solution (10 μL, 0.10 wt %) was placed on the carbon grid, and the excess solution was blotted with filter paper. The sample was dried at room temperature for 24 h. The microscopy image was obtained by using a JEM-2100F microscope (JEOL, Japan) with an accelerating voltage of 200 kV.
spectroscopy, UV−vis spectroscopy, dynamic light scattering, microcalorimetry, and transmission electron microscopy (TEM).
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EXPERIMENTAL SECTION
Materials. Pluronic F127 with an average structure of (ethylene glycol)99−poly(propylene glycol)67−poly(ethylene glycol)99 (BASF) was used as received. Caprolactone, stannous octoate, OCL (MW 1250 Da), and 1,6,-diphenyl-1,3,5-hexatriene were used as received from Aldrich. Toluene (Aldrich) was dried over sodium before use. Synthesis of F-CLs. F-CLs were synthesized by ring-opening polymerization of the caprolactone in the presence of F127.16,19 To synthesize F-CL4 (Table 1), F127 (5.00 g, 0.40 mmol) was dissolved in toluene (80 mL), and the residual water was removed by azeotropic distillation to a final volume of about 20 mL. Caprolactone (0.91 g, 8.00 mmol) and stannous octoate (5 μL, 0.012 mmol) were added to the reaction mixtures. They were stirred at 120 °C for 24 h under the dry nitrogen atmosphere. The polymerization product was purified by precipitation into diethyl ether, and the residual solvent was removed under vacuum. The yield was about 78%. Other polymers with a different OCL block lengths (Table 1) were similarly prepared by varying the amount of caprolactone. 1 H and 13C NMR Spectroscopy. 1H NMR spectra of F-CLs (Table 1) in CDCl3 (500 MHz NMR spectrometer; Varian) were used to determine the composition and block length of the polymers. 13C NMR spectral changes of the F127 (24.0 wt %), F-CL4 (24.0 wt %), and F-CL12 (9.0 wt %) in D2O were compared at 10, 37, and 60 °C. The temperature of each system was equilibrated for 20 min before the measurement. Gel Permeation Chromatography. A gel permeation chromatography system (Waters 515) with a refractive index detector (Waters 410) was used to obtain the molecular weights and molecular weight distributions of the polymers. N,N-Dimethylformamide was used as an eluting solvent. The poly(ethylene glycol)s (PEGs) (Polysciences, Inc.) with a molecular weight range of 200−20 000 Da and polydispersity index (Mw/Mn) of 1.05−1.15 were used as the molecular weight standards. A HR 4E column (Waters) was used. X-xay Diffractometry. The X-ray diffraction data of solid polymers were recorded with a Rigaku INTL2200 diffractometer using Cu Kα radiation (λ = 0.154 nm) at a scanning rate of 1°/min at 20 °C. In addition, polymer gels prepared from polymer aqueous solutions (F127: 24.0 wt %; F-CL4: 24.0 wt %; and F-CL12: 9.0 wt %) at 37 °C were also studied. The polymer aqueous solutions were stored at 37 °C for 20 min to form a gel, and the X-ray diffraction data of the gel were obtained at a scanning rate of 1°/min at 37 °C. Differential Scanning Calorimetry. A differential scanning calorimeter (DSC; SINCO DSC N-650) was used to study the melting temperatures of the polymers in a temperature range of 20−60 °C with a heating and cooling rate of 3.0 °C/min. Polymer (about 5.0 mg) was loaded in a cell, and the heat exchange was recorded during the second heating cycle. Phase Diagram. The sol−gel transition of the polymer aqueous solution was investigated by the test tube inverting method.20 The 4216
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RESULTS AND DISCUSSION End-group modification of Pluronic (F127) was carried out by ring-opening polymerization of caprolactone onto F127. The chemical structure of the OCL end-capped F127 (F-CL) is as follows: H−(OCH2CH2CH2CH2CH2CO)x−[(OCH2CH2)m− (OCH 2 CH(CH 3 )) n −(OCH 2 CH 2 ) m ]−(OOCCH 2 CH 2 CH 2 CH2CH2)x−OH, where m and n of F127 are 99 and 67, respectively. From the 1H NMR spectra of F127 in Figure 1,
DSC thermograms also confirmed the crystalline domain formation of PEG of the F127 and F-CLs (Figure S1b). The melting temperature of the F-CLs decreased from 50.3−56.3 °C to 41.3−47.4 °C as the number of the repeating units of OCL increased from 0 (F127) to 23 (Table 1). The enthalpy of melting also decreased from 105.9 to 56.6 J/g as the number of the repeating units of OCL increased from 0 (F127) to 23 (Table 1). The decreases in melting point and enthalpy of melting indicate the interference of the PEG crystallization process by the end-capped OCL.9,10,24 In particular, the reversal of the magnitude of bimodal melting peaks for F-CL15 and FCL23 suggests that the melting transition of the OCL crystalline domain might be involved in the PEG melting transition of the polymers. To minimize the effect of OCL crystalline domain in studying the self-assembly and phase diagram, F127, F-CL4, and F-CL12 were selected for further detailed studies. The phase diagrams of F127, F-CL4, and FCL12 aqueous solutions determined by the test tube inverting method are shown in Figure 2a. Aqueous solutions of F127
Figure 1. 1H NMR spectra (CDCl3) of the OCL end-capped F127 (FCL4). 1H NMR spectra of F127 and OCL (MW 1250 Da) as purchased are shown for comparison.
the peaks at 1.1−1.2 ppm (−OCH2CH(CH3)−) and 3.3−3.8 ppm (−OCH2CH2− and −OCH2CH(CH3)−) were assigned.8,16 From the 1H NMR spectra of OCL in Figure 1, the peaks at 1.3−1.5 ppm (−CH2CH2CH2CH2CH2COO−), 1.5−1.8 ppm (−CH2CH2CH2CH2CH2COO−), 2.2−2.4 ppm (−CH 2 CH 2 CH 2 CH 2 CH 2 COO−), and 4.0−4.1 ppm (−CH2CH2CH2CH2CH2COO−) were assigned. Therefore, the areas of the 1H NMR spectra at 1.1−1.2 ppm (methyl protons of F127) and 2.2−2.4 ppm (methylene protons of OCL next to carbonyl) were used to calculate the composition and the molecular weight of the F-CLs by the following equations: A 2.2 − 2.4 /A1.1 − 1.2 = 4x /3n = 4x /201
where A1.1−1.2 and A2.2−2.4 are the areas of the peak at 1.1−1.2 and 2.2−2.4 ppm, respectively, in the 1H NMR of F-CL. Based on the assignment, the number of repeating units of the endcapped OCL was calculated to be in a range of 4−23, and the corresponding total molecular weight of F-CLs was calculated in a range of 13 510−17 840 (Table 1). The molecular weight and molecular weight distribution of F-CLs were also determined by gel permeation chromatography relative to poly(ethylene glycol) standards, which were in the range of 11 370−14 810 and 1.2−1.3, respectively (Table 1). F127 is a semicrystalline polymer, with the crystalline phase consisting of PEG and the amorphous phase formed by PPG and PEG.21,22 The characteristic peaks of X-ray diffraction at 2Θ = 19.1° and 23.3° of the polymer indicated that crystalline domain formation of PEGs of both F127 and F-CLs (Figure S1a). The fact that the peak intensity decreased without any change in the peak position suggests that the crystalline structure of F127 is not affected but crystallinity decreased by end-capping by OCL.23 In addition, a small crystallization peak of OCL at 2Θ = 21.7° was shown for F-CL15 and F-CL23.10,24
Figure 2. (a) Phase diagram of polymer aqueous solutions. Circles indicate sol−gel transition temperatures, and stars indicate the gel-tosyneresis transition temperatures determined by visual observation of phase separation between polymer and water. The transition temperatures were reproducible within ±1 °C. (b) Change in modulus (G′) of polymer aqueous solution as a function of temperature. The polymer concentrations were 24.0 wt % (F127), 24.0 wt % (F-CL4), and 9.0 wt % (F-CL12).
underwent a sol-to-gel-to-sol transition as the temperature increased in a concentration range of 15.0−30.0 wt %.6,7 The sol and gel states maintained their transparency over the studied temperature range. However, the F-CL4 aqueous solution underwent a sol-to-gel-to-syneresis transition as the temperature increased, whereas the F-CL12 aqueous solution underwent a gel-to-sol-to-gel-to-syneresis transition as the temperature increased. The phase transition of the F127 4217
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aqueous solution has been intensively studied, where the unimer-to-micelle transition and conformational change in micelle packing have been suggested for the sol-to-gel and gelto-sol transition mechanisms, respectively.17,18 Even though the hydrophobicity of the F-CL4 is greater than F127, the sol−gel transitions were observed at higher concentrations than F127. However, the stronger hydrophobic association of OCL of FCL4 led to the sol-to-gel-to-syneresis transition of the F-CL4 aqueous solution. In particular, the hydrophobic association of OCL was strong enough for F-CL12 to form a gel even at low temperatures. In addition, the concentration range of the FCL12 exhibiting sol−gel transition was lower than F127. The gel melting was observed as the temperature of F-CL12 gel (low temperature gel) increased. As the temperature increased further, a sol-to-gel transition was observed, similar to that of the F127 aqueous solution. As the temperature increased further, the gel-to-syneresis transition was observed by the increased hydrophobic association of the OCL. At concentrations lower than the sol−gel phase transitions being observed, the aqueous polymer systems of F127, F-CL4, and F-CL12 were not strong enough to resist the flow when the test tube was inverted and thus were regarded as a sol state in the investigated temperature range 0−70 °C. At polymer concentrations higher than the sol−gel transition being observed, the polymer aqueous system formed a gel in the same investigated temperature range. The phase transition of the polymer aqueous solutions was studied by the dynamic mechanical analysis in a temperature range of 4−50 °C (Figure 2b). The polymer concentrations were selected as 24.0 wt % (F127), 24.0 wt % (F-CL4), and 9.0 wt % (F-CL12) to show the sol−gel transitions of the polymer aqueous solutions. The gel-to-sol-to-gel transition of F-CL12 and sol-to-gel transition of F127 and F-CL4 accompanied a large change in modulus. The low modulus
