Interactions of Amphiphilic Polyelectrolytes and Neutral Polymeric

Oct 2, 1999 - Masanobu Mizusaki,†,‡ Neil Kopek,‡ Yotaro Morishima,† and. Françoise M. Winnik*,‡. Department of Macromolecular Science, Grad...
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Interactions of Amphiphilic Polyelectrolytes and Neutral Polymeric Micelles: A Study by Nonradiative Energy Transfer Masanobu Mizusaki,†,‡ Neil Kopek,‡ Yotaro Morishima,† and Franc¸ oise M. Winnik*,‡ Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, and Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 Received May 11, 1999. In Final Form: July 28, 1999 The interactions of hydrophobically modified poly(N-isopropylacrylamides) (HM-PNIPAM) and unimolecular micelles formed by fluorescently labeled copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS) and N-n-dodecylmethacrylamide or N-adamantylmethacrylamide were investigated by fluorescence spectroscopy and dynamic light scattering measurements. The HM-PNIPAM copolymers were prepared by free-radical copolymerization of N-isopropylacrylamide (NIPAM) and either N-ndodecylacrylamide or N-(1-adamantylacrylamide). Pyrene- and naphthalene-labeled HM-PNIPAM derivatives were prepared by reaction of 1-pyrenylmethylamine, 4-(1-pyrenyl)butylamine, or 1-(1-naphthyl)ethylamine with copolymers of NIPAM, the hydrophobic monomer, and N-(acryloxysuccinimide). In aqueous solutions, the HM-PNIPAM copolymers form multimolecular micelles ranging in size from 25 to 60 nm (25 °C), as determined by dynamic light scattering and fluorescence measurements based on the extent of nonradiative energy transfer (NRET) between naphthalene and pyrene in mixtures of naphthalene- and pyrene-labeled HM-PNIPAM derivatives. The interactions between HM-PNIPAM and hydrophobically modified PAMPS were studied by NRET measurements in solutions of (1) naphthalene-labeled HMPNIPAM and pyrene-labeled PAMPS and (2) unlabeled HM-PNIPAM and a copolymer of AMPS carrying both naphthalene and pyrene chromophores. Each technique gave evidence for the formation of complexes between PAMPS micelles and HM-PNIPAM chains. The complexation was shown to depend on temperature, the ratio of the two polymers, and the molecular structure of the hydrophobic substituents.

Introduction Water-soluble polymers carrying a small number of hydrophobic substituents have been the focus of intense research over the past decade.1 The interest is generated by the exceptional impact they have on the rheology of aqueous fluids even if only present in very low concentration.2 In commercial products they are brought in contact with other additives, such as surfactants, salts and other polymers. Polyelectrolyte-colloid interactions, in particular, play an important role in industrial processes such as water treatment by colloidal flocculation,3 stabilization of concentrated preceramic suspensions,4 and immobilization and stabilization of enzymes in polyelectrolyte complexes.5 A mechanistic understanding of the interactions among the various components of a formulation is necessary to design more performant fluids. Fundamentally, one needs to consider the competition between forces driving toward intrapolymeric association and those leading to intermolecular hydrophobic interactions between hydrophobic groups of the polymer and those of the † ‡

Osaka University. McMaster University.

(1) For example, see: Hydrophilic Polymers, Performance with Environmental Acceptability; Glass, E. D., Ed.; Advances in Chemistry Series, 248; American Chemical Society: Washington, DC, 1996 and references therein. (2) For example, see: Polymers as Rheology Modifiers; Schultz, D. N., Glass, J. E., Eds.; Advances in Chemistry Series, 462; American Chemical Society: Washington, DC, 1991. (3) Schwoyer, W. L. K., Ed. Polyelectrolytes for Water and Wastewater treatment; CRC Press: Boca Raton, FL, 1981. (4) Cesarano, J., III; Aksay, I. A. J. Am. Chem. Soc. 1988, 71, 1062. (5) Kabanov, V. A. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J. P.; Davies, R. M., Schulz, D. N., Thies, C. M., Eds; Springer-Verlag: New York; 1994; p 151.

colloidal additive. Intrapolymeric micelles may provide a solubilizing environment for the cosolute, thus shifting the equilibrium toward association. However, intrapolymeric micellization often renders the hydrophobic groups less available for interactions with other molecules. In this study, we examined the interactions between a series of hydrophobically modified copolymers of sodium 2-(acrylamido)-2-methylpropane-sulfonate (AMPS) and hydrophobically modified copolymers of N-isopropylacrylamide (NIPAM). The overall solutions properties of these copolymers are summarized in Table 1 and briefly reviewed in the following sections. The macroscopic properties of hydrophobically modified polymers can be traced to the existence of hydrophobic microdomains created by the assembly of hydrophobic segments. Such interactions depend on the detailed architecture of the polymers: on the one hand, the distribution of the hydrophobic groups along the chain, as described by McCormick and co-workers6 and, on the other hand, by the rigidity and bulkiness of the pendent groups, as shown by Morishima and co-workers in their extensive studies of amphiphilic polyelectrolytes.7 The conformation of hydrophobically modified polyelectrolytes in water is controlled by two opposing forces: (1) the attraction of the hydrophobic substituents driven by hydrophobic “forces” created as soon as alkyl groups are brought in contact with water and (2) the electrostatic repulsion between the charged units of the polymer chain. If the (6) Kramer, M. C.; Welch, C. G.; Steger, J. R.; McCormick, C. L. Macromolecules 1995, 28, 5248. (7) Morishima, Y. In Multidimensional Spectroscopy of Polymers: Vibrational, NMR and Fluorescence Techniques; Urban, M. W.; Provder, T., Eds.; ACS Symposium Series, 598; American Chemical Society: Washington, DC, 1995; p 490.

10.1021/la9905721 CCC: $18.00 © 1999 American Chemical Society Published on Web 10/02/1999

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Figure 1. Chemical structures of the polymers used in this study. Table 1. Overall Properties of Hydrophobically Modified Sodium Poly(2-acrylamido-2-methylpropane sulfonates) and Hydrophobically Modified Poly(N-isopropylacrylamides) HM-PAMPS

HM-PNIPAM

micellar properties water solubility temperature sensitivity

intrapolymeric; diameter ) 20-30 nm soluble if x < 50 mol %, x ) hydrophobic group mol % soluble in water at all temperature

ionic strength sensitivity

size affected

interpolymeric; diameter ) 30-60 nm soluble if x < 2 mol %, x ) hydrophobic group mol % soluble in cold water, phase separation occurs for T ≈ 30 °C micelle size insensitive cloud point slightly affected

hydrophobic group content is sufficiently low and if the groups are randomly distributed along the chain, polyelectrolyte chains adopt an extended conformation. As the hydrophobic group content increases, the polyelectrolytes will tend to contract and form highly compact structures consisting of hydrophobic microdomains surrounded by hydrophilic charged chain fragments, known as “unimolecular micelles” or “unimers”. Random copolymers of AMPS and methacrylamides N-substituted with bulky hydrophobic groups exhibit a strong predilection for intrapolymeric association, even in concentrated solutions.8 Copolymers of AMPS and N-adamantylmethacrylamide (PAMPS-Ad, Figure 1) show a particularly strong tendency to form intrapolymeric micelles, while copolymers of AMPS and N-n-dodecylmethacrylamide (PAMPS-Dod, Figure 1) are less prone to intrapolymeric association and exhibit a marked sensitivity to the method of polymer dissolution and to sample history.8,9 Additives may severely disturb the conformation of hydrophobically modifed PAMPS. For example, oppositely charged surfactant micelles have been shown to interact with PAMPS-Dod, yielding mixed polymeric (8) Morishima, Y.; Nomura, S.; Ikeda, T.; Seki, M.; Kamachi, M. Macromolecules 1995, 28, 2874. (9) Hashidzume, A.; Morishima, Y. Unpublished results.

micelles.10 The occurrence and type of PAMPS/surfactant association vary with the hydrophobic content of the polymer and the charge of the micelles.11 Complexes between PAMPS and lysozyme, a basic protein, also form, but only if the solution composition lies within specific pH and ionic strength domains.12 There have been only few studies between PAMPS and neutral colloids. In these systems complexes only form if there exist the possibility of association between hydrophobic moieties of the polyelectrolyte and hydrophobic sites of the neutral species. Neutral surfactant micelles provide one such system;13 hydrophobically modified neutral polymer are yet another example of this situation. The present work examines the associations that occur in mixed solutions of neutral copolymers and hydrophobically modified PAMPS. The neutral copolymers selected for the study are hydrophobically modified poly(N-isopropylacrylamides) (10) Mizusaki, M.; Morishima, Y.; Yoshida, K.; Dubin, P. L. Langmuir 1997, 13, 6941. (11) Mizusaki, M.; Morishima, Y.; Dubin, P. L. J. Phys. Chem. B 1998, 102, 1908. (12) Sato, T.; Mattison, K. W.; Dubin, P. L.; Kamachi, M.; Morishima, Y. Langmuir 1998, 14, 5430. (13) Hashidzume, A.; Mizusaki, M.; Yoda, K.; Morishima, Y. Langmuir 1999, 15, 4276.

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(HM-PNIPAM).14,15,21 When dissolved in water these copolymers form polymeric micelles, which possess the following salient features: (1) they consist of hydrophobic domains surrounded by hydrophilic polymer main chains; (2) they exist in extremely dilute solutions and always involve several polymer chains; (3) they are stable indefinitely in cold water but are severely disrupted when their aqueous solutions are heated above a critical temperature. Poly(N-isopropylacrylamide) itself does not associate in cold water, but its aqueous solutions also undergo thermoreversible phase separation at a critical temperature (31 °C).16 The phase separation mechanism of PNIPAM in water has been shown to take place in two steps:17 (1) the collapse of individual polymer chains from a highly hydrated extended coil into a globule and (2) aggregation of the globules triggering macroscopic phase separation. When solutions of HM-PNIPAM are heated above their cloud point, the micellar structures are disrupted severely: the hydrophobic core is destroyed and the hydrophobic groups are accommodated, mostly as isolated entities, within the separated polymer-rich phase.18 The phase transition temperature depends modestly on the structure of the hydrophobic group, its location along the chain, and its level of incorporation. The hydrophobic substituents of the HM-PNIPAM’s employed in this study were chosen to match those incorporated along the PAMPS chains, namely, the n-dodecyl and the adamantyl moieties. The two alkyl groups have approximately the same number of carbon atoms (12C for n-dodecyl and 10C for adamantyl), but they differ greatly in flexibility and packing ability. In addition to PNIPAM-Dod and PNIPAM-Ad (Figure 1), we prepared also fluorescently labeled polymers, carrying either naphthyl (Np) groups or pyrenyl groups (Py) as well as either dodecyl or adamantyl moieties (Figure 1). These copolymers were selected to enable a study of the HM-PAMPS/HM-PNIPAM association by the photophysical process of nonradiative energy transfer (NRET) between two chromophores.19 The process originates in dipole-dipole interactions between an energy donor in its excited state and an energy acceptor in its ground state. The probability of energy transfer between chromophores depends sensitively on their separation distance and to a lesser extent on their relative orientation. Therefore, in mixed solutions of polymers carrying either energy donor groups or energy acceptor groups, the efficiency of NRET between the two labels can be related to the extent of interpolymeric association. If the two chromophores are attached to the same polymer chain, as in the case of the doubly labeled polyelectrolyte PAMPSDod-Np-Py (Figure 1), the degree of NRET between Np* and Py can be related to the conformation of the polymer. When the polymer adopts an extended conformation, the energy transfer efficiency will be low. In contrast, highly efficient NRET can occur when the polymer adopts a compact globular conformation. In the first part of this paper, we describe the preparation of fluorescently labeled HM-PNIPAM copolymers and (14) Schild, H. G.; Tirrell, D. A. Langmuir 1991, 7, 1319. (15) Winnik, F. M.; Davidson, A. R.; Hamer, G. K.; Kitano, H. Macromolecules 1992, 25, 1876. (16) Heskins, M.; Guillet, J. E. Macromol. Sci., A2 1968, 1441. (17) Tiktopulo, E. I.; Uversky, V. N.; Lushchik, V. B.; Klenin, S. I.; Bychkova, V. E.; Ptitsyn, O. B. Macromolecules 1995, 28, 7519. (18) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992, 25, 7306. (19) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; Chapter 10. (20) Winnik, F. M. Polymer 1990, 31, 2125. (21) Ringsdorf, H.; Venzmer, J.; Winnik, F. M. Macromolecules 1991, 24, 1678.

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their solution properties in water, below and above their cloud point. In the second part, we characterize the complexes formed between HM-PAMPS and HM-PNIPAM in mixed solutions of polymers carrying identical hydrophobic groups and monitor how the interactions are affected by changes in temperature and ionic strength. The micellar characteristics of the complexes in aqueous phase were investigated by fluorescence spectroscopy, dynamic light scattering, and turbidity measurements performed below and above the cloud point of HMPNIPAM. Experimental Section Materials. Analytical grade solvents were used without purification, except when indicated. Water was deionized with a Barnstead NANOpure water purification system. Dioxane was purified by distillation from sodium under nitrogen. N-Isopropylacrylamide (NIPAM) was purified by recrystallization from toluene/hexane 10/1 v/v. 1-Adamantanamine, N-isopropylacrylamide, dodecylamine, triethylamine, acryloyl chloride, AIBN, 1-(1-naphthyl)ethylamine, and 1-pyrenylmethylamine hydrochloride were purchased from Aldrich Chemicals. N-Acryloxysuccinimide (NASI) was obtained from Eastman Kodak Chemicals. 4-(1-Pyrenyl)butylamine hydrochloride20 and N-n-dodecylacrylamide21 were prepared as described previously. The preparation of the modified sodium poly(2-acrylamido-2-methylpropane sulfonates), PAMPS-Dod-Py,22 PAMPS-Dod-Np-Py,22 and PAMPSAd-Py23 was reported previously. Instrumentation and Techniques. 1H NMR spectra were recorded on Bruker 200 or 500 MHz spectrometers. Infrared spectra were recorded on a BioRad FTS-40 spectrometer. UVspectra were measured with a Hewlett-Packard 8452A photodiode array spectrometer equipped with a Hewlett-Packard 89090A temperature controller. Gel permeation chromatography (GPC) was performed with a Waters WISP 700 system equipped with a Waters RI 410 differential refractometer. Four Ultrastyragel columns were used, eluted with THF at a flow rate of 0.8 mL min -1. Dynamic light scattering measurements were performed on a Brookhaven BI9000 AT instrument equipped with an argon laser (λ ) 514 nm, scattering angle 90°). Data were analyzed using the software provided by the manufacturer (CONTIN calculations). Solution viscosities were determined with an Ubbelohde viscometer with solutions of the polymers in THF kept at 27 °C. Cloud points were determined by spectrophotometric changes in turbidity of solutions heated at a constant rate (0.2 °C min-1) in a magnetically stirred UV cell as described previously.20 The cloud point values were taken as the temperature corresponding to a 5% increase in turbidity of the sample. Fluorescence Measurements. Steady-state fluorescence spectra were recorded on a SPEX Fluorolog 212 spectrometer operated by a GRAM/32 data system. Temperature control of the samples was achieved using a water-jacketed cell holder connected to a Neslab circulating bath. The temperature of the sample fluid was measured with a thermocouple immersed in a water-filled cuvette placed in one of the four cell holders. Excitation spectra were measured in the ratio mode. Emission spectra were not corrected. They were recorded with an excitation wavelength of 344 nm (pyrene) and 290 nm (naphthalene). Emission and excitation slit widths were set at 1.0 mm. Solutions in water were not degassed. Solutions in methanol were degassed by a 1-min vigorous bubbling of MeOH-saturated argon through the solutions. Determination of the Spectroscopic Parameters. The pyrene excimer-to-monomer ratios (IE/IM) were calculated by taking the ratio of the intensity (peak height) at 480 nm to the intensity at 376 nm. The extent of pyrene emission due to NRET from naphthalene to pyrene was assessed by the recording the ratio IPy/INp of the emission intensity at 376 nm (pyrene) to the emission intensity at 340 nm (naphthalene). (22) Yamamoto, H.; Mizusaki, M.; Yoda, K.; Morishima, Y. Macromolecules 1998, 31, 3588. (23) Morishima, Y.; Tominaga, Y.; Kamachi, M.; Okada, T.; Hirata, Y.; Mataga, N. J. Phys. Chem. 1991, 95, 6027.

Interactions of Polyelectrolytes and Micelles Sample Preparation. Solutions of HM-PNIPAM used for spectroscopic and dynamic light scattering analysis were prepared from stock solutions (5.0, 2.0, or 1.0 g L-1) kept at 5 °C to ensure complete dissolution of the polymers. Aliquots of the stock solutions were diluted to the desired concentrations. They were kept at room temperature for at least 2 h prior to measurements. Solutions of HM-PAMPS were prepared also from stock solutions (2 g L-1) in water which were sonicated for 3 min at room temperature and kept overnight at room temperature. Mixed solutions were obtained by dilution of polymer stock solutions and, in the case of salt effect studies, aqueous NaCl (2 M). These were allowed to equilibrate for 10 h at room temperature prior to DLS or fluorescence measurements. Synthesis. N-(1-Adamantyl)acrylamide (AdAAm). Acryloyl chloride (5.9 mL, 0.073 mol) was added dropwise at 40 °C to a solution in dichloromethane of 1-adamantanamine (10 g, 0.066 mol), triethylamine (11 mL, 0.079 mol), and 2,6-di-tertbutylcresol (a few mg) kept under nitrogen. When all of the starting material had been consumed (approximately 2 h, as monitored by TLC), the reaction mixture was cooled to room temperature. It was washed with aqueous HCl (0.5 N), aqueous sodium bicarbonate, water, and brine. The organic layer was dried over MgSO4. Evaporation of the solvent gave a solid which was purified by recrystallization from acetone (9.55 g, yield 79%). IR: 3437, 3325, 2973, 2775, 1651, 1540 cm-1. 1H NMR (200 MHz, CDCl3): 1.68 (br s, 6 H), 2.05 (m, 9 H), 5.50 (br s, 1H), 5.65-6.25 (m, 3 H). N-n-Dodecylacrylamine (DodAAm). Acryloyl chloride (4.6 mL, 0.057 mol) was added dropwise at 0 °C to a solution in dichloromethane of n-dodecylamine (10 g, 0.054 mol), triethylamine (10.6 mL, 0.076 mol), and 2,6-di-tert-butylcresol (10 mg) kept under nitrogen. The reaction mixture was stirred overnight at room temperature. It was washed with aqueous HCl (0.5 N), aqueous sodium bicarbonate, water, and brine. The organic layer was dried over MgSO4. Evaporation of the solvent gave a solid, which was purified by recrystallization from THF/n-hexane (6.23 g, yield 48%). IR: 3430, 3302, 2958, 2923, 2853, 1655, 1473 cm-1. 1H NMR (CDCl , 200 MHz): 0.87 (t, 3 H), 1.25 (br s, 18 H), 1.50 3 (m, 2H), 3.34 (q, 2H), 5.50 (br s, 1H), 5.65-6.2 (m, 3H) ppm. Polymerizations. Copolymer of NIPAM and DodAAm (PNIPAM-Dod). A solution of NIPAM (5.06 g, 0.045 mol) and DodAAm (0.052 g, 0.23 mmol) in dioxane (25 mL) was degassed by vigorous bubbling of nitrogen for 30 min. The solution was then heated to 65 °C under nitrogen, and AIBN (0.0105 g, 6.39 × 10-2 mmol) was added to the solution. The reaction mixture was stirred at 65 °C for 24 h. It was cooled to room temperature. The polymer was isolated by precipitation into hexane. It was purified by three consecutive precipitations from THF into hexane (4.36 g, 85%). Copolymer of NIPAM, DodAAm, and N-Acryloxysuccinimide (NASI) (PNIPAM-Dod-NASI). The polymer was prepared by the procedure used to obtain PNIPAM-Dod, starting with NIPAM (4.98 g, 0.044 mol), DodAAm (0.050 g, 0.21 mmol), and NASI (0.079 g, 0.46 mmol): yield 4.12 g, 81%. Copolymer of NIPAM, AdAAm, and N-Acryloxysuccinimide (NASI) (PNIPAM-Ad-NASI). The polymer was prepared by the procedure used to obtain PNIPAM-Dod, starting with NIPAM (5.0 g, 0.046 mol), AdAAm (0.0 53 g, 0.22 mmol), and NASI (0.083 g, 0.47 mmol): yield 4.25 g, 83%. Pyrene-Labeled PNIPAM-Dod (PNIPAM-Dod-Py). Triethylamine (40 mg, 0.32 mmol) was added to a solution of PNIPAM-Dod-NASI (1.050 g) and 1-pyrenylmethylamine hydrochloride (10.0 mg, 0.037 mmol) in THF (7 mL). The solution was stirred at room temperature in the dark for 24 h. The N-isopropylamine (0.36 mL) was added to the reaction mixture, which was stirred for an additional 4-hr period. The polymer was isolated by precipitation in hexane. It was purified by further precipitations from THF into diethyl ether (0.722 g, 69%). Naphthalene-Labeled PNIPAM-Dod (PNIPAM-Dod-Np). This polymer was prepared by the same procedure as PNIPAMDod-Py, using 1-(1-naphthyl)ethylamine and without added triethylamine. Naphthalene-Labeled PNIPAM-Ad (PNIPAM-Ad-Np). This polymer was prepared by the same procedure as PNIPAMDod-Np.

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Figure 2. Synthetic scheme of the preparation of fluorescently labeled hydrophobically modified poly(N-isopropylacrylamides). Pyrene-Labeled PNIPAM-Ad (PNIPAM-Ad-Py). This polymer was prepared by the same procedure as PNIPAM-Dod-Py, using 4-(1-pyrenyl)butylamine, hydrochloride, instead of 1pyrenylmethylamine hydrochloride.

Results Preparation and Characterization of N-Isopropylacrylamide Copolymers. To ensure that the various labeled copolymers have the same molecular characteristics as the respective unlabeled counterparts, they were prepared from a single sample of a reactive polymer, namely a copolymer of NIPAM and N-acryloxysuccinimide (NASI) and either N-n-dodecylacrylamide or N-(1-adamantacrylamide) (Figure 2). The hydrophobic group content was kept low (0.5-1.0 mol %), since a higher level of incorporation leads to polymers insoluble in water. Attachment of the labels to the copolymer backbone was achieved by reacting the purified NASI/NIPAM copolymers with chromophores bearing a short amino-terminated alkyl substituent. Specifically, the copolymers were labeled with naphthalene (Np) by reaction with 1-(1naphthyl)ethylamine. They were labeled with pyrene (Py) by reaction with either 4-(1-pyrenyl)butylamine or (1pyrenyl)methylamine. In all syntheses, unreacted NASI groups were converted to isopropylacrylamide units by reaction with isopropylamine added to the reaction mixture prior to workup (Figure 2). The molecular weights of the copolymers were assessed by viscosity measurements of solutions in THF (Table 2).24 The degree of labeling was determined from the UV absorbance of polymer solutions in methanol, using 4-(1pyrenyl)butylamine (342 nm ) 32 800) and 2-naphthyl(24) The Mark-Houwink equation employed in the calculations (see Table 2) was established for unmodified PNIPAM. It is assumed here that the equation is also valid for HM-PNIPAM. Since the measurements are carried out in THF, the solution properties of the polymers are not expected to be affected significantly by the presence, on the polymer chain, of a small number of hydrophobic substituents.

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Table 2. Composition and Physical Properties of the Polymers composition (mol fraction) polymer PNIPAM-Ad-Py PNIPAM-Ad-Np PNIPAM-Dod PNIPAM-Dod-Py PNIPAM-Dod-Np PAMPS-Dod-Py PAMPS-Dod-Np-Py PAMPS-Ad-Py

Rd 0.45 0.45 1.45 1.15 1.15 50 50 50

Py

Np

0.72 1.48 0.65 0.60 0.96 1.03 1.11

[η] (mL g-1)

[Py] (mol g-1)

mol wt

4.66 ×

000a

55.0 47.3 52.9 35.3 35.3

480 480 000a 571 000a 306 000a 306 000a

5.68 × 10-5 4.47 × 10-5 4.16 × 10-5 4.84 × 10-5

700 000b 35 000c

3.97

10-5

[Np] (mol g-1) 1.31 × 10-4 5.13 × 10-5 1.73 × 10-4

a Measured by viscosity, from [η] ) 9.59 × 10-3 M 0.65 (Fujishige, S. Polym. J. 1987, 19, 297). b Measured by static light scattering v (Morishima, Y., private communication). c Measured by static light scattering, ref 8. d R ) Ad or Dod; see Figure 2.

ethylamine (282 nm ) 6740) as model compounds. Methanol was chosen as a solvent in these quantitative measurements, after it was confirmed that the polymers did not undergo aggregation in this solvent. Moreover the absorbance spectra of the labels in these polymeric solutions were identical in shape to the spectra of the corresponding dyes is the same solvent.25 For the doubly labeled copolymers the pyrene content was determined first; the naphthalene content was then calculated from the absorption at 282 nm, after correction for pyrene absorption at this wavelength. The level of n-dodecyl groups incorporation in PNIPAM-Dod-Np, PNIPAM-Dod, and PNIPAM-Dod-Py was determined from the 1H NMR spectra of the respective copolymers, using the broad singlet at 4.0 ppm due to the resonance of the isopropyl proton of the NIPAM units and the triplet at 0.9 ppm due to the resonance of the terminal methyl group protons of the n-dodecyl substituents. We note that the measured ratio of the two monomeric units was identical, within experimental error, to the monomer feed composition. It was not possible to use 1H NMR data to calculate the degree of adamantyl incorporation in PNIPAM-Ad, because of unfortunate overlaps of the resonances due to the methylene protons of the polymer backbone and those of the adamantyl moieties. We assume here that the adamantyl content of these copolymers is identical to the monomer feed composition, as in the case of the N-ndodecylacrylamide copolymer. Solution Properties of the NIPAM Copolymers in Water. Evidence for Micellar Structure. Even though PNIPAM exhibits many properties characteristic of amphiphilic polymers, it does not form aggregates in water, below the cloud point of the solution. No signal is detected by quasi-elastic light scattering (QELS) analysis of aqueous PNIPAM solutions (20 °C, 1.0-5.0 g L-1). In contrast, a strong signal is detected when the QELS measurements are performed on aqueous solutions of the hydrophobically modified copolymers (20 °C, 1.0-5.0 g L-1). The effective hydrodynamic diameter of the polymeric micelles ranges from 25 to 60 nm (Table 3) depending on the nature of the hydrophobic substituent and the degree of labeling of the polymer. The fact that PNIPAM-Ad-Np forms significantly larger micelles than the other copolymers is surprising, since all polymers have approximately the same molecular weight (Table 2). This may reflect the packing restrictions within the micellar core imposed by bulky adamantyl groups or a higher level of adamantyl group incorporation. As discussed in the previous section, it was not possible to determine accurately by 1H NMR spectroscopy the Ad content of this polymer. Aqueous solutions of the copolymers became turbid when heated, signaling the occurrence of a lower critical solution temperature (LCST). The LCST values of the (25) Winnik, F. M. Chem. Rev. 1993, 93, 587.

Table 3. Properties of the Polymers in Water LCST (°C) polymer

H2O

PNIPAM-Ad-Py PNIPAM-Ad-Np PNIPAM-Dod 29.5 ( 0.2 PNIPAM-Dod-Py 30.0 ( 0.2 PNIPAM-Dod-Np 30.0 ( 0.2 PAMPS-Do-Np-Py PAMPS-Ad-Py PAMPS-Dod-Py

NaCl (0.2 M)

27.5 ( 0.2 27.5 ( 0.2 27.5 ( 0.2

mean diameter (polydispersity) 25 ( 2 nm (0.08) 66 ( 6 nm (0.09) 28 ( 2 nm (0.17) 27 ( 2 nm (0.15) 27 ( 2 nm (0.15) 30 ( 2 nm (0.17) 12.4 nm (ref 8) 29 ( 1 nm (0.13)

copolymers are depressed slightly, compared to the homopolymer (Table 3). The same trend was reported previously for the LCST values of a series of hydrophobically modified PNIPAM’s bearing low levels of n-alkyl chains.21 Spectroscopy of the NIPAM Copolymers in Solution. A Brief Review. The photophysical properties of the pyrene-labeled hydrophobically modified NIPAM copolymers (HM-PNIPAM) prepared for this study follow the same trends as those of similarly labeled HM-PNIPAM reported in detail elsewhere.15,18,21,26 Upon excitation at 344 nm, PNIPAM-Dod-Py in water exhibits an emission due to locally isolated excited pyrene chromophores (intensity IM, pyrene “monomer” emission) with the (0,0) band located at 376 nm, together with a broad featureless emission centered at 480 nm (intensity IE) due to pyrene excimers. Evidence from excitation spectra and time dependent fluorescence measurements indicates that the pyrene excimer emission originates predominantly from preformed ground-state aggregates.25 The pyrene aggregates are believed to contribute to the hydrophobic microdomains created within the polymeric micelles detected by QELS. The pyrene-naphthalene pair of chromophores is known to interact as energy donor (Np) and energy acceptor (Py) by nonradiative energy transfer with a characteristic distance, R0, of 28.6 Å.27 In mixed solutions of pyrene- and naphthalene-labeled polymers excitation at 290 nm, a wavelength at which most of the light is absorbed by Np will result in a complex emission spectrum consisting of the emission of Np* (310-400 nm) and the emission of Py* (370-550 nm) excited by transfer of energy from Np*, if the two chromophores are in close enough proximity to satisfy the separation distance and orientation requirements of NRET. The emission spectrum of a mixed solution of PNIPAM-Dod-Py (0.1 g L-1) and PNIPAM-Dod-Np (0.4 g L-1) upon excitation at 290 nm is shown in Figure 3, together with the spectra of aqueous (26) Yamazaki, A.; Song, J. M.; Winnik, F. M.; Brash, J. L. Macromolecules 1998, 31, 109. (27) Berlman, I. B. Energy Transfer Parameters of Aromatic Compounds; Academic Press: New York, 1973.

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Table 4. Composition of the Mixed Solutions Used in the Energy Transfer Experiments polymers (g L-1) PNIPAM-Dod-Np (0.4) PNIPAM-Dod-Np (0.4) PNIPAM-Ad-Np (0.4) PNIPAM-Dod-Np (0.4) PNIPAM-Dod-Np (0.4) PNIPAM-Ad-Np (0.4) PNIPAM-Dod (0-0.35) PNIPAM-Dod (0-0.36)

PNIPAM-Dod-Py (0.1) PNIPAM-Dod-Py (0.1) PNIPAM-Ad-Py (0.1) PAMPS-Dod-Py (0.05) PAMPS-Dod-Py (0.05) PAMPS-Ad-Py (0.05) PAMPS-Dod-Np-Py (0.05) PAMPS-Dod-Np-Py (0.05)

Figure 3. Fluorescence spectra of solutions in water at 25 °C of PNIPAM-Dod-Np (0.4 g L-1), PNIPAM-Dod-Py (0.1 g L-1), and a mixture of the two polymers (total polymer concentration, 0.5 g L-1); see Table 4 for the relative concentration in the mixture; λexc 290 nm.

solutions of PNIPAM-Dod-Np (0.4 g L-1) and PNIPAMDod-Py (0.1 g L-1). In the spectrum of the mixed solution, a large enhancement of the pyrene monomer emission is observed, together with a modest decrease in the intensity of naphthalene emission. The pyrene excimer emission is not affected by the presence of naphthalene-labeled polymer, indicating that the unassociated pyrene chromophores interact almost exclusively with naphthyl groups. Thus, efficient NRET takes place between Np and Py chromophores linked to different polymer chains, giving strong support for the occurrence of interpolymeric micelles. A qualitative measure of the relative efficiency of energy transfer in mixed polymer solutions at 25 °C can be obtained by taking the ratio IPy/INp of the intensity of the emission at 390 nm to that at 340 nm.28 In this scale a larger value reflects enhanced NRET efficiency. The ratio is slightly larger for aqueous solutions of PNIPAM-AdNp and PNIPAM-Ad-Py (IPy/INp ) 1.1), compared to that measured in the case of mixed solutions of PNIPAM-DodNp/PNIPAM-Dod-Py of identical chromophore and polymer concentration (IPy/INp ) 0.6). Temperature Effects. Mixed Solutions of HMPNIPAM Copolymers. A solution of PNIPAM-Dod-Np (0.4 g L-1) and PNIPAM-Dod-Py (0.1 g L-1), obtained by mixing solutions of each polymer was heated from 15 to 40 °C in the sample holder of the fluorescence spectrometer. Fluorescence spectra were monitored as a function of temperature. A sharp increase of the extent of NRET from Np* to Py occurred as the solution temperature (28) Kramer, M. C.; Steger, J. R.; Hu, Y.; McCormick, C. L. Macromolecules 1996, 29, 1992.

solvent H2O 0.2 M NaCl H2O H2O 0.2 M NaCl H2O H2O 0.2 M NaCl

[Py] mol L-1

[Np] mol L-1

10-6

5.80 × 5.87 × 10-6

2.04 × 10-5 2.04 × 10-5

4.46 × 10-6 4.42 × 10-6

2.03 × 10-5 2.05 × 10-5

2.33 × 10-6 2.27 × 10-6

9.03 × 10-6 8.81 × 10-6

Figure 4. Changes as a function of increasing temperature of the ratio IPy/INp of the intensity of pyrene emission to the intensity of naphthalene emission for solutions in water of mixtures of PNIPAM-Dod-Np and PNIPAM-Dod-Py (full circle) and PNIPAM-Ad-Np and PNIPAM-Ad-Py (open circle); see Table 4 for the polymer concentrations; λexc 290 nm.

reached the macroscopic cloud point. The phenomenon is presented (Figure 4) in terms of the changes in the ratio IPy/INp during heating scans from 15 to 40 °C of mixed solutions. The ratio remains constant as the solution is heated from 15 to 26 °C. Upon further heating it increases sharply to reach a constant value at 32 °C. The midpoint of the transition (29 °C) corresponds to the cloud point determined by turbidity measurements (Table 3). Qualitatively similar results were obtained when a mixed solution of the adamantyl-substituted PNIPAM’s was heated under the same conditions (Figure 4). Energy transfer is more efficient in the phase separated aqueous mixtures of the adamantyl-substituted copolymers (IPy/ INa ) 8) than in mixtures of the dodecyl derivatives (IPy/ INp ) 5), presumably as a consequence of the higher naphthalene content of the PNIPAM-Ad-Np, compared to PNIPAM-Dod-Np. A mixed solution of the n-dodecylsubstituted polymers in aqueous NaCl (0.2 M) was monitored as well. In the presence of salt, the midpoint of the transition assumes a slightly lower value (27 °C). A similar NaCl-induced lowering of the LCST of PNIPAM aqueous solutions has been reported previously, based on the results of turbidity measurements.29,30 Mixed Solutions of HM-PAMPS and HM-PNIPAM. The interactions between HM-PAMPS and HM-PNIPAM were examined by NRET with two types of mixed solutions. In one series of experiments (interpolymeric NRET), the extent of energy transfer from Np* to Py was assessed in solutions of Np-labeled HM-PNIPAM and Py-labeled HMPAMPS carrying identical hydrophobic groups, either (29) Park, T. G.; Hoffman, A. S. Macromolecules 1993, 26, 5045. (30) Inomata, H.; Goto, S.; Otake, K.; Saito, S. Langmuir 1992, 8, 687.

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Figure 5. Fluorescence spectra of solutions in water at 25 °C of PNIPAM-Dod-Np (0.4 g L-1), PAMPS-Dod-Py (0.05 g L-1), and a mixture of the two polymers (total polymer concentration, 0.45 g L-1); see Table 4 for the relative concentration in the mixture; λexc 290 nm.

adamantyl or n-dodecyl. In the other set of experiments (intrapolymeric NRET), we measured the changes in the extent of energy transfer from Np* to Py in the doubly labeled polyelectrolyte PAMPS-Dod-Np-Py as a function of added PNIPAM-Dod. Both systems were investigated also by DLS measurements aimed at probing changes in the size of either the polyelectrolyte unimers or the PNIPAM interpolymeric micelles. In all cases, mixed solutions were prepared from stock solutions in water or aqueous NaCl of each polymer. Interpolymeric NRET Experiments. The emission spectrum of a mixed solution of PAMPS-Dod-Py and PNIPAMDod-Np, excited at 290 nm, is displayed in Figure 5, together with the emission spectra of PNIPAM-Dod-Np and PAMPS-Dod-Py, recorded under identical conditions. The enhancement of the pyrene emission and the decrease of the naphthyl emission in the mixed system signal that the two polymers interact. We note that the extent of NRET between Np* and Py (IPy/INp ) 1.3) is lower in this situation, compared to the value recorded in the case of mixtures of PNIPAM-Dod-Py and PNIPAM-Dod-Np (IPy/INp ) 5.0). As the ionic strength of the mixed solution increases, the energy transfer efficiency decreased slightly, from IPy/INp ) 1.3 in water to IPy/INp ) 0.8 [NaCl] ) 0.2 M. Next, mixed polyelectrolyte/PNIPAM solutions in either water or 0.2 M NaCl were heated from 20 to 40 °C, and their fluorescence spectra were recorded as a function of temperature. A decrease in NRET efficiency occurred as the temperature of the solution reached 26 °C. This change is displayed in Figure 6, where the changes with temperature of the ratio IPy/INp in a mixed solution of PNIPAMDod-Np and PAMPS-Dod-Py are plotted, when the solution is heated from 20 to 40 °C and subsequently cooled to 20 °C. We note that the midpoint of the transition (T ) 29 °C) corresponds to the cloud point of PNIPAM-Dod-Np and that the heat-induced changes are reversible (Figure 6). Thus, the complexes formed between HM-PNIPAM and HM-PAMPS are disrupted as the PNIPAM chains collapse and aggregate. The polymers reassociate as the temperature decreases and the PNIPAM chains uncoil and recover their water solubility. Intrapolymeric NRET Experiments. In this situation, where we observe the spectroscopic properties of the dilabeled polyelectrolyte PAMPS-Dod-Np-Py in the pres-

Mizusaki et al.

Figure 6. Changes as a function of temperature of the ratio IPy/INp of the intensity of pyrene emission to the intensity of naphthalene emission for a solution of PNIPAM-Dod-Np (0.4 g L-1) and PAMPS-Dod-Py (0.05 g L-1) in water upon heating (open circle) and subsequent cooling (full circle).

Figure 7. Changes as a function of PNIPAM-Dod of the ratio IPy/INp of the intensity of pyrene emission to the intensity of naphthalene emission for solutions of PAMPS-Dod-Np-Py (0.05 g L-1) in water: temperature 20 °C; λexc 290 nm.

ence of the unlabeled PNIPAM-Dod, changes in fluorescence properties of the system will reflect perturbations in the polyelectrolyte conformation upon addition of neutral polymer. This technique was found very effective in monitoring the interactions of PAMPS-Dod-Np-Py with nonionic surfactant micelles.13 In water and in 0.2 M aqueous NaCl, PAMPS-Dod-Np-Py adopts a tightly collapsed conformation, as revealed by highly efficient NRET between Np* and Py (IPy/INp ) 12, water).22 The micellar structures formed by PAMPS-Dod-Np-Py under the preparation method employed in this study (see experimental conditions) have an average diameter of 30 nm, as revealed by DLS measurements. This value is larger than that expected for unimer micelles of this polymer (ca. 15 nm)9 and suggests that in our experiments PAMPSDod-Np-Py exists in the aggregate state and not in the unimer state. Upon addition of PNIPAM-Dod to a solution in water of PAMPS-Dod-Np-Py (0.05 g L-1), the extent of energy transfer between Np* and Py decreases with increasing PNIPAM-Dod concentration, as shown in Figure 7, where the changes in IPy/INp are plotted as a function of PNIPAM-Dod concentration. The decrease in IPy/INp indicates a modest perturbation of the PAMPSDod-Np-Py aggregates upon complexation with PNIPAMDod. However the effect detected here is much less pronounced than in mixed systems of PNIPAM-Dod-Np-

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Figure 8. Distributions of apparent diameters for (a) PNIPAM-Dod and (b) mixed solutions in water of PNIPAM-Dod and PAMPSDod-Np-Py (0.4 g L-1) as a function of PNIPAM-Dod concentration. Data were normalized to constant signal intensity for solutions of different concentrations of scattering species.

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Figure 9. Conceptual model of the dynamic association of HM-PAMPS unimers with HM-PNIPAM multimolecular micelles in aqueous solution below and above the cloud point of HM-PNIPAM.

Py and micelles of the neutral surfactant n-dodecyl hexaoxyethylene glycol monoether (C12EO6) reported previously.13 We carried out a study of the effect of solution temperature, from 20 to 40 °C, on the extent of intrapolymeric NRET in PAMPS-Dod-Np-Py (0.05 g L-1) alone and in the presence of PNIPAM-Dod (0.2 g L-1). In both cases, the ratio IPy/INp decreased slightly upon heating, but the changes were insensitive to added PNIPAM-Dod. It has been observed in previous studies of the spectroscopy of PAMPS-Dod-Np-Py that perturbations of the conformation of the polymer chain are often reflected by changes in the ratio I3/I1 of the intensity of the third vibronic band of the pyrene emission to that of the first vibronic band.13 This ratio decreases with increasing polarity of the pyrene environment. We monitored this parameter as a function of PNIPAM-Dod concentration in mixed solutions of PNIPAM-Dod (0-0.5 g L-1) and PAMPS-Dod-Py (0.05 g L-1). The ratio remained constant, I3/I1 ) 0.82, a value indicating that the pyrene label remains confined in the nonpolar hydrophobic core of the unimer micelles. Thus, even though the conformation of PAMPS-Dod-Np-Py is affected by the presence of PNIPAMDod, as evidenced by the NRET measurements, the polarity of the hydrophobic microdomains remains unchanged. Dynamic light scattering measurements were performed on mixed solutions of PNIPAM-Dod and PAMPSDod-Np-Py, keeping the concentration of the polyelectrolyte constant (0.4 g L-1) and adding increasing amounts of PNIPAM-Dod, following an experimental protocol identical to that used in the fluorescence study. All measurements were performed at 20 °C, well below the cloud point of PNIPAM-Dod. The ionic strength of the solutions was kept at 0.2 M. Histograms of the distribution

of the apparent hydrodynamic diameters of PAMPS-DodNp-Py alone and in the presence of increasing amounts of PNIPAM-Dod are presented in Figure 8, together with the histograms recorded with solutions of PNIPAM-Dod in the same concentration domain. Histograms of size distributions recorded from solutions of either PNIPAMDod or PAMPS-Dod-Np-Py indicate that the micellar assemblies formed by the two polymers have similar sizes (∼30 nm). The analysis of mixed solutions of PAMPSDod-Np-Py and PNIPAM-Dod reveals a unimodal distribution of micellar aggregates, which increase from 30 to 48 nm in average diameter with increasing PNIPAM-Dod concentration (0-3.2 g L-1). We note that the average diameter of PNIPAM-Dod micellar assemblies increased from 28 to 37 nm in the same concentration range. Therefore it seems unlikely that the unimodal distribution observed in mixed solutions is due to the fortuitous superposition of two distributions of micellar structure, but rather that it reflects the association of the two copolymers into multipolymeric micelles each consisting of a PAMPS-Dod-Np-Py chain decorated with several PNIPAM-Dod chains. The complexation between the polymers is expected to take place primarily between n-dodecyl substituents. Conclusions In our study of mixtures of the polyelectrolyte HMPAMPS and the neutral amphiphilic polymer, HMPNIPAM, with fluorescence spectroscopy and dynamic light scattering, we have obtained strong indications of the complexation between charged and neutral polymer micelles. A conceptual model of the interactions, derived from the overall results, is depicted in Figure 9. When a neutral micelle encounters an HM-PAMPS micelle, it may

Interactions of Polyelectrolytes and Micelles

first associate to “hydrophobic patches”31 on the surface of the HM-PAMPS unimers. These patches, consisting of clusters of n-dodecyl groups (PAMPS-Dod-Np-Py) or adamantyl groups (PAMPS-Ad-Py) provide a favorable environment for the hydrophobic substituents linked to the PNIPAM chains. This initial association is expected to induce the disruption of the multimolecular micelles of HM-PNIPAM, with subsequent adsorption of several HMPNIPAM chains on the charged micelles. Evidence from NRET points to the fact that the polyelectrolyte conformation is mostly preserved upon complexation: the extent of intrapolymeric NRET in PAMPS-Dod-Np-Py decreases only slightly, in contrast to the effects observed upon addition of neutral surfactant micelles to the same polymer. Moreover, the mixed HM-PAMPS/HM-PNIPAM (31) Morishima, Y. et al. manuscript in preparation.

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micelles dissociate readily as the temperature of the solutions is raised above the LCST of PNIPAM (31 °C). At this temperature the PNIPAM chains collapse and dissociate from the unimers. This dissociation is accompanied by phase separation of aggregated HMPNIPAM’s. The heat induced phase separation is reversible, upon cooling mixed micelles are formed again, as evidence by NRET measurements (Figure 6). Acknowledgment. The work was supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada to F.M.W. and by a fellowship to M.M. from the Ministry of Education, Science, Sports, and Culture, Japan. LA9905721