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Hydrophobic Domains in Thermogelling Solutions of Polyether-Modified Poly(Acrylic Acid) Agnes K. Ho,‡,† Lev E. Bromberg,‡ Paul D. T. Huibers,‡ Andrea J. O’Connor,† Jilska M. Perera,† Geoff W. Stevens,† and T. Alan Hatton*,‡ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Chemical Engineering, University of Melbourne, Victoria 3010, Australia Received July 6, 2001. In Final Form: November 29, 2001 The temperature-dependent aggregation in aqueous solutions of self-assembling graft copolymers of Pluronic (PEO-PPO-PEO triblock copolymers) and poly(acrylic acid) (Pluronic-PAA) has been studied using fluorescence techniques (steady-state intensity and depolarization and fluorescence lifetimes). Aggregates that form above the critical micellization temperatures (CMT) are micelle-like, with substantially dehydrated PPO core and hydrated PEO-PAA corona, and provide the physical cross-linking required for gelation. Unexpected hydrophobic domains have been observed below the CMT which provide an environment more hydrophobic than the micelles to pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH) and are related to the grafting density of the PAA onto the Pluronic copolymers.
Introduction Copolymers of poly(acrylic acid) (PAA) and polyethers such as poly(ethylene oxide) (PEO) or poly(propylene oxide) (PPO) have been used in a wide variety of applications ranging from hydrophilic coatings1 to thickeners and emulsifiers.2-7 While the conformation of PAA in aqueous solutions depends on pH, ionic strength, the presence of multivalent ions, and so forth, the polyethers exhibit solubility in water that depends on a range of critical parameters such as temperature and salt concentration. That is, PEO or PPO chains can undergo a transition from a homogeneous solution of random coils to a microphaseseparated structure if these critical conditions are met.6 As a result, properties of aqueous solutions of graft- or block-copolymers of PAA and polyethers are sensitive to both ionic strength and temperature. Such sensitivity leads to a variety of phase-separated (self-assembled) structures, including micellar aggregates, in the polyetherPAA solutions.6,7 If PAA segments bonded to two or more polyether chains participating in micelles are long enough, concentration regimes can be found in which the polyetherPAA solutions form viscoelastic gels above certain temperatures.7 The gelation temperature (Tgel) corresponds to the critical micellization temperature (CMT),9 which, in turn, depends on the polymer architecture. Varying the length and ratio of the PEO and PPO segments can * To whom correspondence should be addressed. † University of Melbourne. ‡ Massachusetts Institute of Technology. (1) Chen, W.-L.; Shull, K. R. Macromolecules 1999, 32, 6298. (2) Hourdet, D.; L’alloret, F.; Audebert, R. Polymer 1997, 38, 2535. (3) Hourdet, D.; L’alloret, F.; Audebert, R. Polymer 1994, 35, 2624. (4) L’alloret, F.; Hourdet, D.; Audebert, R. Colloid Polym. Sci. 1995, 273, 1163. (5) L’alloret, F.; Maroy, P.; Hourdet, D.; Audebert, R. Rev. Inst. Fr. Pet. 1997, 52, 117. (6) Hourdet, D.; L’alloret, F.; Durand, A.; Lafuma, F.; Audebert, R.; Cotton, J.-P. Macromolecules 1998, 31, 5323. (7) Bromberg, L. In Handbook of Surfaces and Interfaces of Materials; Nalwa, H. S., Ed.; Academic Press: New York, 2001; Vol. 4, Chapter 7. (8) Chen, G.; Hoffman, A. S.; Kabra, B.; Randeri, K. In Poly(ethylene glycol) Chemistry and Biological Applications; Harris, M. J., Zalipsky, S., Eds.; ACS Symposium Series 680; American Chemical Society: Washington, DC, 1997; Chapter 27. (9) Bromberg, L. J. Phys. Chem. B 1998, 102, 1956.
alter the CMT of the copolymers. From this standpoint, the PEO-PPO-PEO copolymers commercially available under the trade names of Pluronic or Poloxamer in a variety of lengths and PPO/PEO ratios are attractive candidates for conjugation with PAA. In such conjugates, the PPO segments would serve as a hydrophobe. PEOPPO-PEO-PAA copolymers can be synthesized by multistep conjugation of monoamino-terminated polyether to PAA via an amide bond.2,8 Alternatively, as has been recently discovered,9,10 the PEO-PPO-PEO-PAA copolymers can be obtained by a single-step free-radical polymerization of acrylic acid with chain transfer to the polyether. In the resulting copolymers, PAA and polyether are conjugated via C-C bonds. As both PAA and Pluronics are considered safe and have been approved by the U. S. Food and Drug Administration as food additives and pharmaceutical ingredients, the copolymers based on PAA bonded with Pluronics (Pluronic-PAA) could be used advantageously in biomedical applications.11 The PluronicPAA copolymers synthesized by Bromberg10 possess characteristically high molecular weights and extreme sensitivity to temperatures. In the semidilute regime (that typically spans 0.01-1 w/v% concentration range12), the Pluronic-PAA aqueous solutions develop into reversible gels with significant elastic moduli13,14 due to the formation of micelle-like aggregates above a well-defined CMT.14-16 Formation of micelles in Pluronic-PAA solutions has been proven by light scattering,9,17 NMR,18 size-exclusion chromatography,9,14 and spin probe techniques.16,18 Our recent SANS study15 has demonstrated that the micelle-like aggregates formed in Pluronic-PAA solutions above the CMT are unusually uniformly distributed. The gel is actually a micellar matrix with scattering centers similar (10) Bromberg, L. Ind. Eng. Chem. Res. 1998, 37, 4267. (11) Bromberg, L. E.; Ron, E. S. Adv. Drug Delivery Rev. 1998, 31, 197. (12) Bromberg, L. Langmuir 1998, 14, 5806. (13) Bromberg, L. Macromolecules 1998, 31, 6148. (14) Bromberg, L. J. Phys. Chem. B 1998, 102, 10736. (15) Huibers, P. D. T.; Bromberg, L. E.; Robinson, B. H.; Hatton, T. A. Macromolecules, 1999, 32, 4889. (16) Bromberg, L. E.; Barr, D. P. Macromolecules 1999, 32, 3649. (17) Bromberg, L.; Orkisz, M.; Roos, E.; Ron, E. S.; Schiller, M. J. Controlled Release 1997, 48, 305. (18) Bromberg, L. E.; Goldfeld, M. G. Polym. Prepr. 1998, 39, 681.
10.1021/la011027e CCC: $22.00 © 2002 American Chemical Society Published on Web 03/12/2002
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Figure 1. A schematic representation of the Pluronic-PAA graft copolymer in the liquid (below the CMT) and gelled (above the CMT) states. Table 1. Properties of the Pluronic Copolymers Used in This Study9,19 polymera
composition
F38 F68 F88 F98 F108 F127
EO42PO17EO42 EO76PO29EO76 EO103PO39EO103 EO118PO45EO118 EO132PO50EO132 EO100PO65EO100
PEO, CPb, average °C MW MPPO % 4700 8400 11400 13000 14600 12600
990 1680 2280 2610 2920 3780
80 80 80 80 80 70
>100 >100 >100 >100 >100 >100
HLBc >24 >24 >24 >24 >24 18-23
a F stand for flakes. b Cloud point in aqueous 1 wt % solution, °C. c Hydrophilic-lipophilic balance.
in structure, but somewhat smaller than micelles of the Pluronic component, with a dehydrated PPO core of radius 30 Å surrounded by a hydrated PEO corona of outer radius 60 Å. A schematic representation of the polymer in the liquid (below the CMT) and gelled (above the CMT) states is given in Figure 1. Our previous studies concentrated on Pluronic-PAA copolymers that were specifically fractionated after synthesis.14,16 However, as prepared, these copolymers may contain significant amounts of a permanently cross-linked fraction. In the present study, we elucidated the relationship between the unique synthetic route (random grafting of PAA on multiple polyether segments) and the capacity of the resulting copolymers to solubilize hydrophobic solutes in water. The behavior of both unfractionated polymers and fractionated ones devoid of gelled fraction was studied. The present study builds on our previous understanding of the Pluronic-PAA copolymers16 through an analysis of the properties of aggregates formed in their solutions by fluorometric techniques. Solubilizing gel-like domains that are more hydrophobic than the micelles have been discovered, which persist even into the low-temperature regions where no intermolecular micellar aggregates are found. Experimental Section Materials. Pluronic surfactants were a generous gift from BASF Corp. and were used without further treatment. The majority of the work was completed using F127, although other Pluronic copolymers were used in the study on the effect of PPO segment length on the gelling properties of the graft copolymers synthesized here. The properties of all the Pluronic copolymers used in this study are collected in Table 1. (19) 19.BASF Performance Chemicals. Pluronic and Tetronic Surfactants; BASF Corporation: North Mount Olive, New Jersey, 1996.
Poly(ethylene oxide) (PEO, average MW 3400), poly(propylene oxide) (PPO, average MW 3000), acrylic acid (99%), pyrene (99%), and 1,6-diphenyl-1,3,5-hexatriene (DPH, 98%) were all obtained from Aldrich Chemical Co. and were used as received except for pyrene which was repeatedly recrystallized from absolute ethanol following sublimation. Poly(sodium acrylate) (Mw 1.36 × 106) was obtained from American Polymer Standards Co. and was dialyzed against deionized water and lyophilized prior to use. Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-g-poly(acrylic acid) (CAS #186810-81-1) was synthesized by dispersion/emulsion polymerization of acrylic acid along with simultaneous grafting of the resulting poly(acrylic acid) onto the Pluronic backbone as described in detail elsewhere.10 The polymer consisted of 45% Pluronic F127 and 55% poly(acrylic acid) (PAA). The abbreviation Pluronic-PAA is used for the polymer. All other chemicals, gases , and organic solvents of the highest purity available were obtained from commercial sources. Procedures. To prepare solutions of Pluronic-PAA, the polymer samples were dispersed in distilled water and gently stirred at 4 °C for 48 h. The pH was adjusted to 7.0 ( 0.1 with 5 M NaOH as needed. The solutions were filtered through Acrodisc nylon filters (Gelman Sciences) with pore diameters of 0.8 µm, deoxygenated by nitrogen flow and stored at 4 °C. The weight fraction of macroscopic gel particles was measured9 at 15 °C by filtering 1 w/v% Pluronic-PAA through weighted Acrodisc nylon filters (Gelman Sciences) with pore diameters of 0.8 µm. The parameter G,% ) 100x(weight of filtered fraction)/ (weight of initial suspension) was measured for each suspension at least five times. Standard errors for G values obtained for different batches of the copolymer did not exceed 17%. Some of the Pluronic-PAA samples were fractionated in aqueous buffers at 15 °C by size exclusion chromatography (SEC) as previously described.14 In a separate series of experiments, fractions with molecular weight above 7 × 106 were removed, while the lower molecular weight Pluronic-PAA fractions were combined, lyophilized, and reconstituted in deionized water (pH 7.0). These samples were used for a comparative study with the original nonfractionated polymer. Weight-average molecular mass of the Pluronic-PAA samples typically exceeded 5 × 105 Da, and polydispersity of the fractions subjected to SEC varied from 2.3 to 6.7. Fluorescence spectra were recorded on a TimeMaster Fluorescence Lifetime Spectrometer (Photon Technology International, PTI, Canada) in a steady-state mode (band-pass,1.5 nm; integration time, 2 s) using a FeliX software for data acquisition and analysis. Alternatively, a Shimadzu Model RF-5301 PC spectrofluorophotometer with a UV-vis polarizer was used under controlled temperature conditions (slit widths of 1.5 nm) at rightangle geometry. Properties of the aggregates formed in the polymer solutions were assessed by steady-state pyrene or DPH fluorescence studies. A stock solution of 1 mM pyrene in absolute methanol or acetone was prepared, from which 1-3 µL were added to an aerated aqueous sample resulting in a 0.6 µM pyrene concentration. The sample was then allowed to equilibrate for
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at least 20 min at a given temperature, and emission (λex)335 nm) spectra were recorded. The ratio of the intensities of the first (373 nm) to the third (384 nm) vibronic peaks (I1/I3) in the emission spectra of the monomer pyrene was used to estimate the polarity of the pyrene microenvironment, the smaller this ratio the more hydrophobic the environment of the pyrene.20Each spectrum was obtained by averaging three scans, corrected for scatter using an equivalent blank polymer solution, and I1/I3 values were averaged over three different estimates. To load polymer solutions with DPH, 0.1 mL of 0.25 mM DPH solution in methanol was added to the stock polymer solution to a final DPH concentration of 2.5 µM and the resulting mixture was gently stirred for 4 h to reach equilibrium. Emission spectra were recorded between 400 and 460 nm (λex)355 nm), with a band-pass of 8 nm and integration time of 5 s. Polarized steady-state fluorescence intensities of DPH were recorded on a TimeMaster Fluorescence Lifetime Spectrometer (PTI). The fluorescence anisotropy (r) was calculated from
r ) (Ivv - gIvh)/(Ivv + 2gIvh)
(1)
where h and v denote the horizontal and vertical orientations of the excitation and emission polarizers, respectively, and g ) Ihv/ Ihh is an instrumental correction factor measured in a separate experiment and used then to correct all subsequent experimental data. Fluorescence lifetime measurements were carried out on a LaserStrobe Fluorescence Lifetime Spectrometer (PTI), using a TimeMaster software for data acquisition and analyses. The “pulse method” (repetitive excitation with brief pulses of light) was used to excite the sample, and the fluorescence decay was measured using the “stroboscopic method” (reconstruction of the time-resolved decay after a large number of pulses have been sampled, each for a different time following the excitation pulse21). A PTI model PL2300 nitrogen-pulsed laser of picosecond pulse width was used as an excitation source. The scattering solution consisted of powdered nondairy creamer dissolved in water. The scattering wavelength was set at the excitation wavelength for N2 (337 nm) while the emission wavelength was set at 429 nm for DPH (probe concentration 0.5 µM throughout). Other experimental parameters were start delay at 52 ns, end delay at 90 ns, 200 channels, integration time 10 µs, and frequency of laser firing 10 Hz. Neutral density filters from Oriel Instruments (Stratford, CT) were used to control the emission intensities so that high intensities could be obtained without the effects of saturation. To test the goodness of the fit between the measured and calculated fitting function R(t), the following parameters were used: χ2 (0.9 e χ2 e 1.2 for a good fit; χ2 ) 1.0 for a perfect fit), the Durbin-Watson parameter (DW > 1.70, 1.75, 1.80 for single, double, and triple exponential fits, respectively), and the runs test Z (Z > -1.96 for a satisfactory fit with 95% confidence). The fitting functions, and hence the fluorescence lifetimes, were accepted only if all three parameters satisfied the above criteria simultaneously. The rotational correlation time (τφ) was calculated from22
ro/r ) 1 + 3τ/ τφ
(2)
where ro is the limiting value of r for steady-state fluorescence in rigid media where no rotation occurs, and τ is the fluorescence lifetime. In this work, ro)0.362, the DPH anisotropy in propylene glycol at -50 °C,23,24 was adopted.
Results and Discussion Pyrene Fluorescence Measurements. Pyrene has been used widely as a probe of molecular organization in self-assembled systems such as micelles and micro(20) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039. (21) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1986. (22) Perrin, F. J. Phys. Radium 1926, 7, 39. (23) Shinitzky, M.; Barenholz, Y. J. Biol. Chem. 1974, 249, 2652. (24) Shinitzky, M.; Inbar, M. J. Mol. Biol. 1974, 85, 603.
Figure 2. Temperature dependency of the ratio of the intensities of the first to the third vibronic peaks in the emission spectrum of pyrene (I1/I3) in aqueous solutions. Results are shown for water, 1 and 2 w/v% aqueous solutions of Pluronic F127, a physical blend of poly(acrylic acid) (0.55 w/v%) and Pluronic F127 (0.45 w/v%), and for 0.5, 1, 2, and 4 w/v% aqueous solutions of Pluronic-PAA. Concentration of pyrene was 0.6 µM and pH was 7.0 throughout. Unless noted otherwise, PluronicPAA designates copolymers of Pluronic F127 and poly(acrylic acid).
emulsions25-28 and thermally responsive polymers. 29 This molecule is unique as a probe because its fluorescence spectrum has a vibronic fine structure with strong solvent dependence20,30 such that the ratio I1/I3 of the intensities of the first (λ1 ≈ 373 nm) and the third (λ3 ≈ 384 nm) vibronic bands is a sensitive measure of the polarity of the solvating environment for the pyrene, decreasing with increasing hydrophobicity. Pyrene, having low water solubility, is expected to partition preferentially to the most hydrophobic regions in a self-organized system in solution and thus provides a measure for both the presence and the hydrophobicity of such regions. Figure 2 shows the effect of temperature on the I1/I3 ratio in aqueous solutions of Pluronic F127, Pluronic-PAA, and a physical blend of PAA and Pluronic F127. The results for pyrene in deionized water (pH 7.0) shown for comparison are in good agreement with the literature values of 1.87 at ambient temperature reported by Dong et al.30 and 1.95 and 1.92 at 20 °C and 25 °C, respectively, as determined by Nivaggioli et al.31 The characteristic slight decrease in intensity ratio with temperature in water is attributed to temperature-induced changes in the solvent polarity.31 In Pluronic F127 solutions, the I1/I3 ratio falls just below that for water at the low temperatures, indicating a strongly polar environment, but begins to decrease more sharply as the temperature is raised above about 20 °C because of the partitioning of the pyrene to a more hydrophobic environment as pre-micellar aggregates begin to form. The effect of the F127 concentration on the onset of this decrease reflects the mass action law governing the aggregation process. At temperatures above the critical micellization temperatures (CMTs) of 27 and 25 °C for the 1% and 2% solutions, respectively, the process of true (25) Gra¨tzel, M.; Thomas, J. K. Modern Fluorescence Spectroscopy 2; Plenum Press: New York, 1976. (26) Zana, R. In Surfactant Solutions. New Methods of Investigation; Zana, R., Ed.; Marcel Dekker: New York, 1987. (27) Kalyanasundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press: New York, 1987. (28) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Yu, V.; Yaroslavov, A. A.; Kabanov, V. A. Macromolecules 1995, 28, 2303. (29) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992, 25, 7306. (30) Dong, D. C.; Winnik, M. A. Can. J. Chem. 1984, 62, 2560. (31) Nivaggioli, T.; Alexandridis, P.; Hatton, T. A.; Yekta, A.; Winnik, M. A. Langmuir 1995, 11, 780.
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micellization begins and the pyrene reports to hydrophobic PPO-rich micelle cores,32 at which point the environment sensed by the probe is independent of the F127 concentration. The addition of partially neutralized PAA to the Pluronic solution does not change the general features of this behavior but does reduce the I1/I3 ratio significantly to values below 1.4 throughout the temperature range investigated, consistent with published reports that the intensity ratio for PAA solutions (in the absence of other polymers) is around 1.45-1.59, depending on ionization16,33 Thus, the increase in the overall polarity and charge distribution within the solution on the addition of PAA drives the localization of the pyrene to hydrophobic domains, which must be related to the conformation of the PAA in solution. Following the onset of micellization of the Pluronic F127 copolymers, the intensity ratio is further depressed, indicating that the PAA has a strong effect on the polarity even within the Pluronic PPO cores themselves. Apparently, the PAA influences the structure of the micelles, perhaps participating in the micelle formation directly such that the core is composed of both PPO and undissociated PAA segments which may be more hydrophobic than the PPO itself, consistent with reports of complex formation between Pluronic copolymers and PAA because of hydrogen bonding, even at neutral pH.34 It was anticipated that similar behavior would be observed for the Pluronic-PAA copolymers, but this was not the case. The behavior of Pluronic-PAA solutions, where PAA is covalently bound to the Pluronic F127, is very different from that of the blends of Pluronic and PAA polymers in solution at the same relative Pluronic/PAA concentrations. The I1/I3 ratio shows a reversed trend, where it falls below the values for the physical blend at the lower temperatures, but increases on formation of micellar-like aggregates to values approaching but still lower than those for the pure Pluronic micellar solutions at the higher temperatures. This striking result indicates the presence of domains more hydrophobic than the micelles in the Pluronic-PAA solutions, and it can be inferred that as the micelles begin to form, the pyrene partitions preferentially to the PPOrich micellar cores rather than to the hydrophobic domains. This could be a reflection of a higher capacity of the micelles relative to the other hydrophobic domains for the probe in these systems. In contrast to the Pluronic F127 case, there is a strong effect of concentration over the whole temperature range, and higher copolymer concentrations lead to domains that are more hydrophobic, suggesting that there is some increasing aggregation of these domains with increasing concentration or that the concentration averaged intensities, and their ratio, are dominated more by the contribution of the solubilized component as more of it reports to the larger number of hydrophobic domains available at the higher concentration. Added insight into the nature of hydrophobic domains was obtained by fractionating the Pluronic-PAA polymer into low and high molecular weight fractions, below and above 7 × 106, respectively, using size-exclusion chromatography.14 The species of the Pluronic (F127)-PAA copolymer studied herein did not contain any measurable amount of the gel phase that could be filtered (i.e., G ) 0%; see Experimental section for details). The presence of regular short-chain branching characteristic of the graftcomb structure has been observed in fractionated Pluronic(32) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. J. Am. Oil Chem. Soc. 1995, 72, 823. (33) Philippova, O. E.; Hourdet, D.; Audebert, R.; Khokhlov, A. R. Macromolecules 1997, 30, 8278.
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Figure 3. Temperature dependency of the I1/I3 intensity ratio of pyrene in 1 w/v% aqueous solutions (pH 7.0) of PluronicPAA, before and after fractionation. Data points for 1% F127, F127/PAA blend, 1% copolymer “as prepared”, and 1% F127PAA fractionated are given by open circles, open squares, filled circles, and crossed squares, respectively.
PAA by size-exclusion chromatography.14 The behavior of the lower molecular weight component was markedly different from that of the dense, strongly cross-linked higher molecular weight fraction, as shown in Figure 3. The fractionated lower molecular weight component of the Pluronic-PAA in aqueous solutions behaved more like a physical blend of PAA and Pluronic in that the I1/I3 ratio decreased upon micellization to have the same limiting values at the higher temperatures, suggesting that the micelles in the lightly grafted and physically blended polymer solutions are similar in structure and properties. At the low temperatures, however, while the I1/I3 ratio was slightly higher than for the unfractionated polymer, it was still significantly lower than for the physical blend, consistent with the presence of strongly hydrophobic domains even in the fractionated material. These results suggest that the effect of the PAA on the micelle core properties within the high molecular weight gelled fraction is less significant than in the Pluronic/PAA blend owing to the more restricted range of motion of the PAA in this highly cross-linked fraction, that is, the constraints imposed on the more heavily cross-linked system change the nature of the micelle-like domains in the gelled phase. These pyrene fluorescence studies have pointed to the existence of unexpected hydrophobic domains in the Pluronic-PAA copolymers over the entire temperature range investigated here, that is, in both the liquid and gelled states of the Pluronic-PAA solutions, and to the fact that these domains are different from the domains afforded by the cores of the micelles that form only at the higher temperatures. It is still unclear as to the precise reasons for this behavior, but some insight into the nature of these domains is gained in the next two sections on the basis of the results of other fluorescence probing techniques and of an appreciation of the mechanistic aspects of the Pluronic-PAA graft copolymer synthesis. DPH Fluorescence Measurements. DPH (1,6diphenyl-1,3,5-hexatriene) fluorescence can also be used to characterize hydrophobic domains in aqueous solution. The absolute intensity of DPH fluorescence increases with the hydrophobicity of the environment; in water and other polar solvents DPH is quenched and exhibits essentially no fluorescence.27,35 DPH has several favorable properties as a probe of membrane fluidity36 and has been used to (34) Chun, M.-K.; Cho, C.-S.; Choi, H.-K. J. Appl. Polym. Sci. 2001, 79, 1525. (35) 35. Fluorescence Spectroscopy; Wolfbeis, O. S., Ed.; SpringerVerlag: Berlin, 1993. (36) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978, 515, 367.
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Figure 4. Temperature dependency of the relative emission intensity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in Pluronic F127 and Pluronic-PAA solutions (pH 7.0). The intensity was measured at 400 nm (λex)355 nm). The DPH concentration was 4.5 µM.
investigate micellization in Pluronics28,32 and aggregation in thermally responsive polymers.29 DPH fluorescence changes with temperature in selfassociating Pluronic F127 and Pluronic-PAA solutions as shown in Figure 4. The F127 solution shows no significant fluorescence below the CMT and strong fluorescence above the CMT, as has been observed previously for PEO-PPOPEO triblock copolymer solutions.37 The two curves for F127 have essentially the same shape, but there is an approximately 3 °C displacement of the 2% F127 curve to the left relative to the 1% F127 curve. The strong DPH fluorescence at all temperatures for the Pluronic-PAA solutions reveals the presence of a hydrophobic microenvironment for DPH, both above and below Tgel, in agreement with the pyrene experiments. The DPH fluorescence intensity for the 2% Pluronic-PAA solution is higher than that for the 1% solution over the entire temperature range, reflecting a greater overall capacity for the probe at the higher polymer concentration. Knowing that the total DPH concentration is constant at 2.5 µM and that only the DPH in the hydrophobic environments fluoresces, we can use these data to estimate the partition coefficient for the distribution of DPH between the hydrophobic domains and water. The total probe concentration is
Ctot ) φCpol + (1 - φ)Caq ) [φΚ + (1 - φ)]Caq (3) where K ) (Cpol/Caq)eq is the partition coefficient for the equilibrium distribution of the solute between the two phases, φ is the weight fraction of polymer in solution, and Cpol and Caq are the solute concentrations per unit mass of polymer and aqueous phase, respectively. The measured intensity is
Itot ) φCpolIhd + (1 - φ)CaqIaq = φΚIhdCaq
(4)
where Ihd is the probe emission intensity in the hydrophobic domains of the polymer phase per unit concentration in this phase, and it is assumed that the probe emission intensity in water, Iaq, is essentially zero. Eliminating Caq from both equations, and inverting, we obtain
1/Itot ) 1/(IhdCtot) [1 + {(1 - φ)/φK}]
(5)
Therefore, at any given temperature, a plot of the reciprocal intensity versus (1 - φ)/φ should yield a straight line with intercept (IhdCtot)-1 and slope (IhdCtotK)-1, from (37) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414.
Figure 5. Partition coefficient (K) values for DPH in the Pluronic-PAA copolymer solutions. For details, see text.
which the partition coefficient can be readily found by taking the ratio of these two quantities. We show the estimated partition coefficient values for DPH in the Pluronic-PAA copolymer solutions in Figure 5. The solid symbols are the results obtained directly at the indicated temperatures; clearly in the temperature range from about 20 °C to 30 °C, the values (shown by solid diamond symbols) deviate quite markedly from those at the lower and higher temperatures. The reason is that these results fall within the transition region over which pre-micellar and micellar aggregates are beginning to form, and at any given temperature the aggregation states of the 1% and 2% polymer solutions are very different. This is particularly evident for the F127 solutions, where the 1% solution curve is shifted to the right by about 3 °C relative to the 2% curve; this indicates that the relative state of the 2% solution at any given temperature is similar to that of the 1% solution at a temperature 3 °C higher. Such a concentration-dependent transition zone is not as strongly evident from the DPH fluorescence intensities reported here, although it has been noted in the pyrene fluorescence intensity ratio studies reported above, and results on the rotational correlation times for the probe in a range of different environments (discussed below) also show that there is a transition region in the PluronicPAA solutions as in the F127 solutions. To apply eq 5 in this region, then, we should compare the results at equivalent aggregation states rather than at the same temperature. Thus, we have taken the datum point for the 2% solution at temperature T, plotted it with the datum for the 1% solution at a temperature of T + 3 °C, and used the slope and intercept of the resulting line connecting the two points to estimate the partition coefficient. The values thus obtained, shown as the open symbols in Figure 5, are now consistent with the results obtained for the temperatures outside this transition region and indicate a partition coefficient ranging from 100 to 200 as the temperature increases from 10 °C to 40 °C. These values are 103-fold lower than those reported for pyrene in Pluronic-PAA micelles.16 Such pyrene/DPH selectivity can be explained by the varying location of the probes: pyrene is fully buried within the hydrophobic PPO cores of the micelles, while the linear, rigid DPH molecule tends to be situated on the interface between the hydrophobic core and the water-rich PAA corona.7,16,18 It must be recalled that the partition coefficient is based on the total polymer mass and not on the fraction of the polymer that provides the hydrophobic domains, which is thought to be quite small; thus, actual partition coefficients for DPH between the hydrophobic domains themselves and water may be at least an order of magnitude or two higher than what we report here. It is also difficult to interpret the partition coefficient at the higher temperatures where there are two solubilizing domains presentsthe hydrophobic do-
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Figure 6. Fluorescence lifetimes of DPH in Pluronic F127 and Pluronic-PAA solutions as a function of temperature overlaid with similar data for a range of solvents.
mains and the micellar coressas the derivation of the equation assumed that the specific emission intensity for the DPH was the same in both types of environment, which may not be true. At this stage, therefore, we cannot ascertain what the relative distribution of the probe is between the two domains. Other information on the environment sensed by the probe can be obtained using complementary fluorescence measurements, however, such as the fluorescence lifetime and the fluorescence rotational correlation time, which are discussed below. The fluorescence lifetime of the probe reflects its interaction with the environment and the stabilization conferred by that environment on the excited state. In Figure 6 the fluorescence lifetimes of DPH in Pluronic F127 and Pluronic-PAA solutions are compared with those obtained in water, acetone, 25% PEO, and PPO. It is evident that the less polar the environment, the greater the stability of the excited fluorophore and that in nonassociating solvent environments there is little effect of temperature on the fluorescence lifetime of the probe. For the 2% F127 solution at temperatures below the CMT, however, the fluorescence lifetime increases with increasing temperature, probably reflecting an increased interaction of the probe with pre-micellar aggregates as they form and grow. At and above the CMT the fluorescence lifetime is almost invariant with temperature and coincides directly with the lifetimes observed in pure PPO at these temperatures, consistent with our understanding that the probe is solubilized within a PPO-rich micellar core. For the 2% Pluronic-PAA solution, however, the fluorescence lifetimes are temperature invariant both below and above the CMT, although there appears to be a slight increase at this transition temperature. These data are representative of those for all the graft copolymer concentrations studied, that is, no effect of the polymer concentration on the fluorescence lifetime was observed, and in most cases there was no discernible change in the lifetime at the CMT either. In all cases, the lifetime was longer than that observed in the F127 solution, indicating a more stabilizing environment for the probe in the Pluronic-PAA suspension. This observation is consistent with the I1/I3 results obtained for pyrene, which indicated a less polar environment for the solute in the PluronicPAA solutions than observed for Pluronic block copolymer micelles themselves. The fluorescence studies reported above point to significant solubilization of the DPH in hydrophobic domains over the whole temperature range. Further insight into the nature of these domains can be gained by considering the effects of the solubilization environment on the rotational diffusion rates of this probe, which provide an indication of the relative restriction to movement, that is, the microviscosity, afforded by the different environments.
Figure 7. Rotational correlation time (τφ) of DPH as a function of solution temperature for (a) PEO and PPO homopolymers, water, and acetone; (b) Pluronic F127 solutions; and (c) PluronicPAA solutions. The results for water, acetone, and PPO are repeated on each of the figures to provide reference points.
The degree to which emitted light is depolarized relative to the polarization of the excitation beam is a direct consequence of this rotational motion of the probe during the lifetime of the excited state of the probe molecule and can be quantified in terms of the rotational correlation time, τφ. In a series of panels to ensure clarity, Figure 7 shows τφ as a function of solution temperature for PEO solutions and pure PPO, for Pluronic F127 and PluronicPAA solutions, and for water and acetone. The results in Figure 7a show that τφ is very small for acetone and somewhat higher for water, consistent with the relative viscosities of these two solvents. In PEO solutions, the resistance to rotational diffusion increases as the polymer concentration is increased from 5% to 25% and the probe experiences an increasingly more restricted environment. The rotational diffusion in pure PPO is significantly slower yet, as anticipated, and, as for the other solvents, its rate increases (τφ decreases) with increasing temperature as a result of increasing thermal motion. In all cases, the drop in τφ with temperature is monotonic. The effect of Pluronic F127 concentration on τφ is shown in Figure 7b. As with the PEO solutions, at the lower temperatures the rotational diffusion rates slow as the F127 concentration is increased, although the concentra-
Hydrophobic Domains in Thermogelling Solutions
Figure 8. Rotational correlation time (τφ) of DPH at a temperature of 12.9 °C as a function of aqueous polymer concentration for solutions of Pluronic F127, Pluronic-PAA, and PEO.
tion effect is significantly stronger than in the PEO case (see discussion of Figure 8 below). With increasing temperature, however, characteristic minima corresponding to the CMTs at the respective concentrations can be discerned. Clearly, upon formation of the micelles, τφ increases owing to an increasing microviscosity of the DPH environment as the micellar cores become more dominated by the PPO blocks of the triblock copolymers. With further increases in temperature beyond about 5-10 °C above the CMT, where the micelles are fully formed and there are no more significant compositional changes in the micelle cores, τφ again decreases owing to the decreasing microviscosity of these micellar cores with increasing thermal motion of the polymer chains in the core. At the higher temperatures, the rotational correlation times for the Pluronic solutions are close to those for pure PPO at these temperatures, suggesting complete solubilization of DPH in essentially dehydrated micellar cores at temperatures significantly above the CMT, again consistent with our understanding of the structure of these micelles. In the Pluronic-PAA solutions (Figure 7c), however, very different behavior is observed. The rotational correlation time is essentially independent of the polymer concentration at low temperatures, and the results only deviate from one another once the micelles begin to formsthe change in the slope of the τφ curve with increasing temperature occurs at earlier temperatures (essentially the CMTs) for the higher polymer concentrationssand indicate the formation of an increasingly more restrictive micellar-like environment for the probe in much the same way as for the F127 micelles. Indeed, the 4% PluronicPAA results approach the PPO curve asymptotically with increasing temperature, as in F127, while the curves for the lower concentrations appear as though they would do so at sufficiently higher temperatures than those investigated here. These results indicate that the solubilizing environment for the probe is the same at low temperatures for all polymer concentrations and are in accord with our earlier conclusions that the hydrophobic domains are not related to changes in aggregation state with polymer concentration at these temperatures but are an intrinsic property of the polymers themselves. The depolarization experiment results for the three different polymers at a temperature of 13 °C (well below their respective CMTs) are summarized as a function of the polymer concentrations in Figure 8. Again, clearly the hydrophobic domains in the Pluronic-PAA solution provide a consistent and almost constant resistance to the rotational motion of the probe over a wide polymer concentration range, while the nonassociating PEO and
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F127 solutions afford increasing impedance to the rotational diffusion as their concentrations are increased. The concentration scales for the PEO and F127 solutions differ by a factor of 5, and with this compression of the PEO scale, the PEO and F127 data are almost indistinguishable. This suggests that the probe senses a restriction to rotational movement in the F127 solution that is equivalent to the restriction provided by the PEO at a 5-fold higher concentration and hence that the F127 coils with which the DPH interacts are more collapsed than the PEO coils at the same concentration. One last observation is that the anisotropy values, r, used in Equation 2 to calculate τφ ranged from 0.07 to 1.5 for the Pluronic-PAA, depending on polymer concentration and temperature. These values are all higher than those for sodium dodecyl sulfate (0.07) but lower than for selfassembling block copolymers with rigid glassy cores (0.19 to 0.27).38 This observation is consistent with results showing that the release rate of solutes from the PluronicPAA micelles is faster than from the glassy cores of micelles of hydrophobically modified polyelectrolytes but is much slower than from low molecular weight surfactants.39 Liquid-State Hydrophobic Domains. The above results all point to the unexpected existence of hydrophobic domains in the liquid state of the Pluronic-PAA solution, which were not observed in our dynamic light scattering17,40 and small-angle neutron scattering15 measurements, in which we established that micelle-like aggregates comparable to triblock copolymer micelles form in the hydrogel state but that no comparably large scattering centers are observed in the liquid state. Different nonmicelle-like hydrophobic domains have been found in similar polymers, it is true, but under different solution conditions. It has been observed, for instance, that PAA/ PEO mixtures will complex through hydrogen bonding at low pH,3 and similar behavior has been seen in Pluronic/ PAA mixtures.41 Chen and Thomas demonstrated that poly(methacrylic acid) can provide a hydrophobic environment for pyrene at low pH, and it is suggested that such behavior is characteristic of poly(acrylic acid) as well.42 These results can be attributed to the loss of ionization by acid groups at low pH. The Pluronic-PAA solutions differ from these examples in that the polyelectrolyte component is highly charged, since at pH 7 the PAA component should be ionized (the pKa of acrylic acid monomer is 4.26 and approximately 6.0 for cross-linked poly(acrylic acid)), and intrachain Coulombic repulsion should force the polymer matrix into an expanded state. This begs the question, then, as to what is the true nature and origin of the hydrophobic domains in the liquid state. One possibility is that small domains are created by the grafting process. Supporting this thesis is the suggestion that, in general, polymers of different composition are incompatible with each other and tend to separate when closely associated.43 In block copolymers, different types of polymers are forced to associate locally through covalent attachment. This can lead to interesting and unique properties not seen in physical mixtures of polymers (which may phase separate) or random copolymers (which lose the characteristics of the pure compo(38) Lee, S. C.; Chang, Y.; Yoon, J.-S.; Kim, C.; Kwon, I. C.; Kim, Y.-H.; Jeong, S. Y. Macromolecules 1999, 32, 1847. (39) Bromberg, L.; Magner, E. Langmuir 1999, 15, 6792. (40) Ho, A. K.; Bromberg, L. E.; O’Connor, A. J.; Perera, J. M.; Stevens, G. W.; Hatton, T. A. Langmuir 2001, 17, 3538. (41) Cole, M. L.; Whateley, T. L. J. Colloid Interface Sci. 1996, 180, 421. (42) Chen, T.; Thomas, J. K. J. Polym. Sci. 1979, 17, 1103. (43) Graham, N. B. In Hydrogels in Medicine and Pharmacy. II Polymers; Peppas, N. A. Ed.; CRC Press: Boca Raton, FL, 1987.
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nents). It is hypothesized here that the PAA graft causes a “folding” of the PPO or PEO or a deionization of the PAA near the graft, creating a pocket large enough to accommodate a pyrene or DPH molecule in a hydrophobic environment, a thesis that is consistent with the unique mechanism of the Pluronic-PAA synthesis.9 It has been established9,10 that the free radicals (R•) generated via heat-induced decomposition of initiators in the presence of both acrylic acid (A) and PEO-PPO copolymers can initiate polymerization of acrylic acid:
R• + nA f R - An• Simultaneously, the free radicals produce macroradicals because of hydrogen abstraction from the polyether:
where X is CH3 or H. Recombination of the Pluronic-radical and poly(acrylic acid)-radical results in the Pluronic-PAA copolymer. Similarly, the Pluronic-PAA (along with the PAA homopolymer) ensues from the chain transfer reaction
The reactivity of the Pluronic-radicals in chain transfer reactions leads to the appearance of the cross-linked or permanently gelled structures:
which is in agreement with other observations that both recombination of the Pluronic-radicals and hydrogen abstraction reactions with participation of free-radical initiators will result in (PEO-PPO-PEO)x homopolymers and gels.44 We wished to minimize the formation of such cross-linked Pluronic-PAA gels and therefore optimized the reaction conditions through testing a range of combinations of initiators.10 Nevertheless, some of the waterinsoluble, cross-linked fraction always results, especially at advanced stages of conversion when above 90% of all acrylic acid monomer has been consumed and macroradicals cause additional grafting onto existing PluronicPAA structures. At high degrees of conversion, a dramatic increase of molecular weight has been observed,9 with the entire system tending to become a gel cluster,45 as predicted by the Flory theory.46 The removal of the gel clusters results in an increase in the I1/I3 ratio (Figure 3), suggesting that these clusters contribute significantly to the nonpolar environment observed for the pyrene in the as-prepared Pluronic-PAA copolymers at temperatures below the CMT. The grafting of PAA to the PPO block is more favorable than to the PEO block, and clearly the number of graft points on a Pluronic copolymer will depend on the length (44) Topchieva, I. N.; Momot, I. G.; Ivanova, V. P.; Efremova, N. V. Moscow University Chem. Bull. 1990, 45, 95. (45) Bromberg, L. Ind. Eng. Chem. Res. 2001, 40, 2437. (46) Flory, P. J. Principles of Polymer Chemistry, 15th ed.; Cornell University Press: Ithaca, 1992.
Figure 9. Effect of the length of the PPO segment (NPPO) on the onset of gelation (Tgel), intensity ratio (I1/I3), and the gel content (G) in 1 w/v% Pluronic-PAA solutions (pH 7.0). Graft copolymers of Pluronics F38, F68, F88, F98, and F108 and PAA were used. Standard errors for G (e17%) are omitted for clarity.
of the PPO block in this copolymer. By the same token, the greater the number of graft points on a given polymer the more restricted will be its ability to participate in multipolymer aggregates, while the number of graft points on a given length of the PPO block in the Pluronic component of the grafted copolymer will determine the degree of cross-linking of any given Pluronic copolymer. These two properties of PPO prompted a study on the effects of the length of the PPO segment in the Pluronic on the properties of the resulting Pluronic-PAA. Pluronics with the same content (80%) of PEO (Table 1) were chosen for this study. Each Pluronic was copolymerized with PAA (0.45:0.55 weight ratio) by the same methodology.10 The properties of the resulting 1% aqueous solutions as functions of the length of PPO segment (NPPO) in the original Pluronic are presented in Figure 9. The ratio I1/I3 at 15 °C, the temperature of gelation, and the content of gelled phase (G) all correlated strongly with NPPO, while the I1/I3 ratio was reciprocally proportional to the content of the gelled phase (R2 > 0.97, cross plot of the data from Figure 9 is not shown). These observations lead to the conclusion that the amount of cross-linking and hence the graft density of the PAA on the Pluronic blocks within the Pluronic-PAA play a significant role in the overall hydrophobicity of the hydrophobic domains at T < CMT. It is also clear from the I1/I3 ratios for the fractionated and unfractionated materials shown in Figure 3 that the higher graft density of the high molecular weight fraction leads to more hydrophobic domains than observed with the low molecular weight fractions. Such domains may be labile and exchange polymer chains with the interchain aggregates (micelles) above the CMT.39 Conclusions The PEO-PPO-PEO-g-PAA (Pluronic-PAA) copolymers investigated here provide unique characteristics that can be exploited in a wide range of applications including topical and internal drug delivery administration, cosmetic formulation, and separation processes.7 These polymers gel reversibly and without phase transitions with changes in temperature, the gelation being caused by an entropically driven self-association of the PPO groups to form micelles that provide the physical cross-linking points necessary for gelation. One of the advantages of these systems stems from their ability to solubilize different hydrophobic materials and subsequently to release them. The nature of the solubilizing environments, and how they compare with those found in conventional Pluronic block copolymer micelles, has been investigated using a range of fluorescence techniques that are often exploited in the
Hydrophobic Domains in Thermogelling Solutions
study of temperature-dependent aggregation in aqueous solutions of self-assembling copolymers; many of the findings were unexpected and point to the existence of hydrophobic domains that are unrelated to the typical domains found in the conventional micelles. While in conventional Pluronic copolymer solutions hydrophobic domains are observed only when micelles are present, that is, above the CMT, it appears that two types of aggregates exist in the Pluronic-PAA solutions. Large micelle-like aggregates form above the CMT, with a substantially dehydrated PPO core and hydrated PEOPAA corona, as with conventional Pluronic systems; it is these aggregates that are responsible for the gelation of the solutions.15 At temperatures below the CMT, however, hydrophobic domains in the Pluronic-PAA copolymers are observed that are not present in the Pluronic solutions. These domains appear to be smaller and more hydrophobic than the larger, micelle-like aggregates in the hydrogel state. Pyrene I1/I3 emission intensity ratios and DPH fluorescence intensity and depolarization patterns show that there is a distinct difference in the environment to which the probes are exposed depending on whether the
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polymer solution is in the liquid or gelled state. These domains are assumed to be created by the process of grafting the poly(acrylic acid) onto the triblock copolymer. The presence of small hydrophobic domains in the liquid form of the gel has implications for the use of the polymer as a slow release matrix, as additional hydrophobic domains will enhance solubilization of drugs, as well as impede diffusion out of the polymer solution or hydrogel. Acknowledgment. AH is grateful for the Postgraduate Overseas Research Experience Scholarship from Melbourne University, which made this work possible. We also would like to thank the Australian Research Council International Researcher Exchange Progamme for financial support. The authors are thankful to Dr. Aleksander Siemierczuk (Photon Technology International, Ontario) for useful discussions on various aspects of this work, in particular with the fluorescence lifetime measurements. LA011027E
