Langmuir 1999, 15, 4185-4193
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Fluorometric and Ultraviolet-Visible Absorption Study of Poly(methacrylic acid) Shells of High-Molar-Mass Block Copolymer Micelles† Miroslav Sˇ teˇpa´nek, Kla´ra Podha´jecka´, and Karel Procha´zka* Department of Physical and Macromolecular Chemistry & Laboratory of Specialty Polymers,‡ School of Science, Charles University in Prague, Albertov 2030, 128 40 Prague 2, Czech Republic
Yue Teng and Stephen E. Webber* Department of Chemistry and Biochemistry & Center for Polymer Research, University of Texas at Austin, Austin, Texas 78712 Received August 31, 1998. In Final Form: February 23, 1999 Polyelectrolyte behavior of the inner part of poly(methacrylic acid) shell and microscopic properties of the interface between the hydrophobic core and the hydrophilic shell in polystyrene-block-poly(methacrylic acid) micelles were studied by UV-vis spectroscopy and by the steady-state and time-resolved fluorometry using two fluorescent probes, 5-(N-octadecanoyl)aminofluorescein (OAF) and 5-(N-dodecanoyl)aminofluorescein (DAF). Two almost identical copolymer samples of polystyrene-block-poly(methacrylic acid), PS-PMA and PS-DPA-PMA, were used in the study, except that the latter was fluorescently tagged between blocks by a pending p-vinyl-9,10-diphenylanthracene group (DPA), which allows also for studying the OAF binding in the interfacial region by means of nonradiative energy transfer from DPA to fluorescein headgroups of either OAF or DAF. Pronounced shifts in pKa of both OAF and DAF, which were observed after binding to polymeric micelles, are explained by a competition of two effects. The first effect is caused by the association processes of amphiphilic probes in the bulk aqueous solution, and the second one is due to an additional energy that is needed for the proton dissociation from the fluorescein -OH group in the anionic polyelectrolyte shell and for the consequent proton transfer into the bulk (the latter is similar to that described by Fromherz et al.).
Introduction In our recent studies of the behavior of dilute solutions of water-soluble polystyrene-block-poly(methacrylic acid) micelles, we have used 5-(N-octadecanoyl)aminofluorescein, OAF, to probe properties of polyelectrolyte shells formed by poly(methacrylic acid), PMA.1 Amphiphilic OAF belongs to the class of lipophilic fluorescent probes that have been developed primarily for membrane studies in biochemistry.2a,b These lipophilic probes may be successfully used also to probe properties of either inorganic surfaces2c or surfactant micelles and various nanostructures formed by synthetic polymers.2d While most results of our fluorometric measurements on OAF bound to polyelectrolyte micelles with PMA shells were consistent with general knowledge of micellar systems and easily understandable, some findings were surprising and difficult to explain. Therefore we have performed a supplementary and more detailed research on the polyelectrolyte behavior of the PMA shell and OAF binding to the high molar mass polyelectrolyte micelles. Despite the fact that * To whom correspondence should be sent. † Presented at Polyelectrolytes ’98, Inuyama, Japan, May 31June 3, 1998. ‡ Supported by Ministry of Education of the Czech Republic (Grant No. VS 97 102).
(1) (a) Sˇ teˇpa´nek, M.; Krijtova´, K.; Procha´zka, K.; Teng, Y.; Webber, S. E. Colloids Surf., A 1999, 147, 79. (b) Krijtova´, K.; Sˇ teˇpa´nek, M.; Procha´zka, K.; Webber, S. E. J. Fluoresc. 1998, 8, 21. (2) (a) Zlatanov, I. V.; Foley, M.; Birmingham, J.; Garland, P. B. FEBS Lett. 1987, 222, 47. (b) Commerford, J. G.; Dawson, A. P. Biochem. J. 1988, 249, 89. (c) Dutta, A. K.; Salesse, C. Langmuir 1997, 13, 5401. (d) Chatenay, D.; Urbach, W.; Messager, W.; Langevin, D. J. Chem. Phys. 1987, 86, 2343.
hundreds of studies with various fluorescein-based derivatives are published every year and the literature on xanthene dyes is so extensive that it seems futile to give specific references, systematic data on the solution behavior on fluorescein-based surfactants with long aliphatic chains in a broad region of pH and ionic strength are missing. To our knowledge, behavior of OAF has not been investigated and described in the whole region of conditions relevant for studies of nanostructures formed by amphiphilic block copolymers. It was the reason why we have also studied properties of OAF in aqueous solutions and in systems of surfactant micelles in a broad region of pH, ionic strengths, and probe concentrations. Block copolymer samples containing a long and strongly hydrophobic block, such as polystyrene, and a long polyelectrolyte block, such as poly(methacrylic acid), do not dissolve in aqueous buffers since water is too strong a precipitant for the hydrophobic part. However, multimolecular spherical micelles consisting of compact cores formed by hydrophobic blocks and protective shells formed (3) Tuzar, Z. ;Webber, S. E.; Ramireddy, C.; Munk, P. Polym. Prepr., Am. Chem. Soc., Div. Polym. Chem. 1991, 32, 525. (4) Tian, M.; Quin, A.; Ramireddy, C.; Webber, S. E.; Munk, P.; Tuzar, Z.; Procha´zka, K. Langmuir 1993, 9, 1741. (5) (a) Procha´zka, K.; Kiserow, D.; Ramireddy, C.; Tuzar, Z.; Munk, P.; Webber, S. E. Macromolecules 1992, 25, 454. (b) Kiserow, D.; Procha´zka, K.; Ramireddy, C.; Tuzar, Z.; Munk, P.; Webber, S. E. Macromolecules 1992, 25, 461. (c) Procha´zka, K.; Martin, T. J.; Munk, P.; Webber, S. E. Macromolecules 1996, 29, 6518. (d) Sˇ teˇpa´nek, M.; Krijtova´, K.; Procha´zka, K.; Teng, Y.; Webber, S. E.; Munk, P. Acta Polym. 1998, 49, 96. (e) Sˇ teˇpa´nek, M.; Krijtova´, K.; Limpouchova´, Z.; Procha´zka, K.; Teng, Y.; Munk, P.; Webber, S. E. Acta Polym. 1998, 49, 103.
10.1021/la981129d CCC: $18.00 © 1999 American Chemical Society Published on Web 04/24/1999
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4186 Langmuir, Vol. 15, No. 12, 1999 Chart 1
by polyelectrolyte blocks may be prepared indirectly by stepwise dialysis in dilute aqueous media.3 Micellar cores are in a nonequilibrium kinetically frozen state in aqueous media (indirect ultracentrifugation4 and fluorometric data5b suggest that, e.g., polystyrene cores are almost glassy) and properties and thermodynamic stability of aqueous micellar systems depend strongly on the polyelectrolyte behavior of shells.5b,c,f
Chart 2
(SAFE). A reconvolution procedure was used to get the true fluorescence decays that were further fitted to multiexponential functions using the Marquardt-Levenberg nonlinear leastsquares method.8 Low values of the χ2 and random distributions of residuals were used as criteria of the fit. Potentiometric Measurements. The pH measurements were performed using a PHM 93 reference pH meter, Radiometer, Denmark, equipped with a combined glass microelectrode PHC 2406.
Results and Discussion
(a) Materials. Copolymer Samples. Amphiphilic polystyreneblock-poly(methacrylic acid) diblock copolymer, PS-PMA, and a corresponding fluorescently tagged copolymer, PS-DPA-PMA, were synthesized by Dr. T. J. Martin at the University of Texas at Austin by means of anionic polymerization. Details on the preparation and the sample characterization are given in ref 6. Structure and characteristics of both samples are given in Chart 1. Polymeric micelles from PS-PMA, micellar molar mass, (Mw)mic ) 8.1 × 106 g/mol, hydrodynamic radius, RH ) 35 nm (in an alkaline buffer, pH 9.3, ionic strength, I ) 0.15), and micelles from PS-DPA-PMA, (Mw)mic ) 8.0 × 106 g/mol, hydrodynamic radius, RH ) 35 nm (in an alkaline buffer, pH 9.3, ionic strength, I ) 0.15), were prepared by stepwise dialysis from 1,4-dioxanewater mixtures into aqueous buffers. The preparation procedure, characterization of polystyrene-block-poly(methacrylic acid) micelles and their general characteristics are given elsewhere.5d Sodium dodecyl sulfate, puriss., Sigma, USA, was used as purchased. Fluorescein, for fluorescence measurements, Fluka, Switzerland, was used as purchased. 5-(N-Dodecanoyl)aminofluorescein and 5-(N-octadecanoyl)aminofluorescein were purchased from Molecular Probes, USA, and used as obtained. The structure of both probes is shown in Chart 2. (b) Experimental Techniques. UV-vis Absorption Spectroscopy. UV-vis absorption spectra were carried out with a Hewlett-Packard 8452a diode-array spectrophotometer. Steady-State Fluorometry. Steady-state fluorescence spectra were recorded with a SPEX Fluorolog 2 fluorometer as described elsewhere.7 Time-Resolved Fluorometry. The time-correlated single photon counting technique was used for measurements of fluorescence lifetimes. The time-resolved fluorescence decays were recorded on a ED 299 T time-resolved fluorometer, Edinburgh Instruments, Inc., equipped with a nanosecond coaxial discharge lamp filled with hydrogen at 0.5 atm (half-width of the pulse ca. 1.2 ns).5d The apparatus allows for a multiplexing regime of the simultaneous acquisition of fluorescence and excitation profiles
(a) Behavior of Free OAF and DAF Probes in Aqueous Solutions. UV-vis Absorption Spectra. As mentioned above, fluorescence behavior of free probes in aqueous solutions was studied firstly to get detailed knowledge of spectral properties in both amphiphilic probes and information necessary for the correct interpretation of their spectra after penetration and sorption in the innermost part of the polyelectrolyte shells of polymeric micelles. Both probes are fluorescein-based pH indicators and their pH-dependent UV-vis absorption and fluorescence spectra are reminiscent of those of low-molarmass fluorescein derivatives. However, due to the surfactant nature of OAF and DAF, both spectra are strongly affected by association processes. In fluorometric studies, concentration of the probe is usually lower in orders of magnitude than the critical micelle concentration, cmc, and any changes in the absorption spectra or selfquenching of the fluorescence due to the probe-micelle formation do not come into account. Nevertheless, surfactants containing long aliphatic chains (with more than 12 methylene groups) are known to form reversible dimers and oligomers at very low concentrations far below the cmc.9 Association processes for ionic surfactants depend strongly on the ionic strength of the solution. Increasing concentration of small ions in the solution screens electrostatic repulsion between fluorescent head-groups and facilitates association of aliphatic chains. The onset of association processes occurs therefore at lower probe concentrations as compared with the salt-free solutions. For the studied probes, the important fluorescent forms of OAF and DAF are dianions with dissociated carboxylic and phenolic groups. In this case, small ions affect not only the electrostatic repulsion between molecules but also dissociation of electrolyte groups. An increase in ionic strength (i) promotes dissociation of fluorescein groups and improves solubility of the hydrophilic part of the probe and (ii) screens the electrostatic repulsion between ionized groups. As concerns the association processes, both effects
(6) Eckert, A. R.; Martin, T. J.; Webber, S. E. J. Phys. Chem. 1997, 101, 1646. (7) Sturtevant, J. L.; Webber, S. E. Macromolecules 1989, 22, 3564.
(8) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (9) Mukerjee, P. J. Phys. Chem. 1965, 60, 2821.
Experimental Section
Polyelectrolyte Behavior of the PMA Shell
Figure 1. Absorption spectra of 3 µM DAF at various pH values. Insert: Absorption spectra of 3 µM fluorescein at various pH values.
compete with each other, which results in a fairly complex behavior of aqueous OAF solutions. The pH-dependent UV-vis absorption spectra of fluorescein, DAF, and OAF for buffer solutions with the ionic strength I ) 0.15 and a constant concentration, cF ) 3.3 × 10-6 mol/L are shown in Figures 1 and 2. For the sake of a fast and smooth discussion of the pH effect on DAF and OAF spectra, we briefly outline general features of the fluorescein behavior (see the insert in Figure 1). Fluorescein exists in aqueous solutions mainly in its quinoid form as a cation, HF+, a neutral molecule, F, monoanion, F-, and dianion, F2-, depending on the pH of the solution. In aprotic solvents, e.g., in acetone, fluorescein exists almost exclusively in its lactone form that is nonfluorescent and absorbs far in the UV region. Literature dissociation constants describing ground-state equilibria between individual species (from left to the right), given by their negative logarithms, are pK1 ) 2.14, pK2 ) 4.45, and pK3′ ) 6.80, respectively.10 Dissociation constants without a prime describe equilibria between the neutral form (expressed as a sum of both neutral species) and the pertinent ionic forms. The absorption band with the maximum at 490 nm corresponds to dianion, F2-, and a band around 445 nm to monoanion, F-. At low pH, the band close to 436 nm corresponds to the neutral fluorescein, F. In the studied pH region, concentration of the protonated cation is negligible and HF+ does not influence the spectra. At high pH 7-12, only the equilibrium between the mono- and dianion is effective and an isosbestic point is obviously located at 464 nm. At lower pH, both dissociation equilibria have to be taken into account simultaneously and the spectra are more complex. However, even in the pH region close to pK2, the position of the maximum of the F2- absorption is pH-dependent. Fluorescein is known to form nonfluorescent H-dimers and aggregates with the blue-shifted absorption bands.2c,11 However for the fluorescein concentrations considered herein, the diffusion-controlled formation of dimers does not occur. The shape and the position of the absorption band of the DAF dianion (Figure 1) at high pH are similar to that of fluorescein, except that the maximum is slightly red(10) (a) Mchedlov-Petrosyan, N. D. Zh. Anal. Khim. 1979, 34, 1055. (b) Leonhardt, H.; Gordon, L.; Livingston, R. J. Phys. Chem. 1971, 75, 245. (11) (a) McRae, E. G.; Kasha, M. J. Chem. Phys. 1958, 28, 721. (b) Kasha, M.; Rawls, H.; El-Bayoumi, M. Pure Appl. Chem. 1965, 11, 37.
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Figure 2. Absorption spectra of 3 µM OAF at various pH values. Insert: Absorption spectra of 3 µM DAF and OAF at pH 10.6.
shifted by ca. 2 nm. The most striking difference is the strong suppression of the monoanion band close to 450 nm, which is almost absent in the measured spectra. At pH 6.5, the long wavelength edge of the spectrum starts to shift to longer wavelengths and at pH 5.5 a welldeveloped maximum appears at 510 nm. The red shift is due to the hydrophobic association of DAF at low pH. The monoanion and the neutral forms of DAF are less soluble than the dianion, and their aliphatic parts associate. Since the hydrophobic association of aliphatic chains is the driving force for the formation of DAF oligomers, a fraction of fluorophores in associates remain relatively far from each other and do not form H-dimers. They experience slightly less polar microenvironment (with a slightly decreased proton donation power), and their spectrum is red-shifted.12 At pH below 3, intensity of the absorption band at 510 nm decreases and a new maximum develops at 480 nm. We assume that it is the red-shifted band of the neutral dye trapped in hydrophobic domains formed by aliphatic chains in DAF oligomers. When comparing the fluorescein and DAF spectra, it is necessary to keep in mind that the aliphatic chain in DAF is attached via an amino-carbonyl bridge which modifies slightly the spectroscopic properties of the dye. Absorption spectra of aqueous OAF solutions are shown in Figure 2. They differ significantly from those of both fluorescein and DAF. The maximum of the dianion OAF band is shifted to the red by ca. 2 nm as compared with fluorescein. The most striking difference is the broadening of the spectra to both shorter and longer wavelengths in the whole pH region (see Figure 2 insert which compares OAF and DAF spectra at high pH). The broadening indicates significant association of OAF aliphatic chains in a broad pH region, and not only at low pHs as it was observed for DAF. Since the formation of OAF associates is a result of hydrophobic interaction of aliphatic parts of amphiphilic OAF molecules, the distances between the fluorescein head-groups in individual associates are fairly random and differ significantly. As a consequence, a fraction of suitably (i.e., closely) located fluorophores form H-dimers with the blue-shifted absorption (see the pronounced shoulder around 470 nm). The remaining fluo(12) (a) Martin, M. M. Chem. Phys. Lett. 1975, 35, 105. (b) Xanthene dyes exhibit a blue shift in solvents with increasing polarity, contrary to a number of common simple dyes. The changes in the spectra are caused by the protonation of the dye. The proton-donation power increases generally with the polarity of the proton-donating solvents and the slightly misleading term “the polarity-dependent shift” is still widely used to discuss the changes in the xanthene dyes spectra.
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Figure 3. Fluorescence intensity, IF, vs ionic strength, I, of 3 µM OAF at various pH values. Insert: Fluorescence intensity, IF, vs concentration, cF, of 3 µM DAF and OAF, pH 10.5.
rophores, which are located far from each other and cannot form H-dimers, experience slightly less polar environment, and their absorption is red-shifted as compared with that for an aqueous solution of nonassociated OAF molecules. The formation of H-associates is obvious in a broad pH region up to very high pHs, despite the fact that the OAF solubility improves with increasing pH. Double-charged fluorescein head-groups repel each other; however their increased concentration in the high pH region promotes association. Very interesting spectra are observed in slightly acid solutions. The probability of dianionic headgroup association is suppressed due to low concentrations of dianions in this pH region, and the nonassociated dianions experience a less polar microenvironment which is manifested by well-pronounced red-shifted maxima close to 504 nm. At pHs around 2, a new band corresponding to the neutral dye appears which is slightly blue-shifted with respect to the DAF spectra. Fluorescence Measurements. Additional information on OAF association may be obtained from fluorescence measurements of DAF and OAF in alkaline solutions (pH 10.5). Fluorescence intensity IF (excitation at 490 nm, emission at 520 nm) as a function of probe concentration, cF, is shown in Figure 3, insert, for a series of solutions with a constant ionic strength, I ) 0.15. While that of DAF is basically a linear function of DAF concentration in the region of cF ca. 10-7 to 10-6 mol/L, fluorescence intensity of OAF increases only in the region of very low concentrations ca. 10-7 mol/L and levels off for slightly higher concentrations due to the aggregation-induced selfquenching. The absolute fluorescence intensity of OAF and also the transition region, in which the fluorescence levels off, depend strongly on I. Effect of the ionic strength on fluorescence intensity is shown in Figure 3 for solutions differing in pH (cF ) 3.3 × 10-6 mol/L). Fluorescence intensity decreases strongly with increasing I, mainly in the region of low I and high pH. Corresponding intensities for DAF do not depend on I and are not shown. Dependencies of fluorescence intensity, IF, at 520 nm on pH (normalized by the maximum intensity (I max F , for pH 12) are shown in Figure 4 for two aqueous solutions of DAF and OAF. Curves for DAF do not depend on concentration and coincide. It is evident from Figures 3 and 4 that curves for DAF are affected by ionic strength and probe concentration only little, while those for OAF depend strongly on both cF and I. The most important features of the fluorescence behavior of OAF are consistent with the above outlined scheme that was used for the discussion of the
S ˇ teˇ pa´ nek et al.
Figure 4. Normalized fluorescence intensity, IF/I max F , vs pH, of DAF (curve 1), 0.3 µM OAF (curve 2), and 3 µM OAF (curve 3).
UV-vis absorption. Association of OAF aliphatic chains is more important at higher concentrations. At high pH, the relative concentration of dianions increases, which promotes their association with all other molecular forms of the probe and leads to self-quenching of the fluorescence. However, the repulsion between ionized head-groups hinders the association and a close approach of dianions and shifts the equilibrium back toward monomer chains. The electrostatic repulsion may be screened very efficiently by small ions in the solution and is the reason why the decrease in the fluorescence intensity with increasing salt content is most pronounced at high pH. A considerable shift in the apparent excited state dissociation constant of OAF (i.e., pKa*′ ) 8.05 for 0.3 µM and 8.95 for 3 µM OAFscurves 2 and 3 in Figure 4) to higher values as compared with both fluorescein (the literature value pKa*′ ) 6.9010b) and DAF (pKa*′ ) 6.55) and the appreciably lower slope of the fluorescence vs pH plot for the higher concentration of the probe (curve 3 in Figure 4) do not reflect the true decrease in the acidity of the excited OAF. Both effects are indirect results of the self-quenching that is more evident at high probe concentration and at high pH: The absolute fluorescence intensity of OAF (cF ) 10-6 mol/L) is more than 10 times lower due to the self-quenching than that of the DAF solution with the same concentration. The complexity of the behavior, which is affected both by the dissociation of ionic head-groups and by the hydrophobic association of aliphatic chains, precludes quantitative analysis of the multiple association equilibria based on the spectroscopic properties of OAF only. Nevertheless, the present experimental study allows us to outline the reasonable qualitative explanation of fluorometric measurements from aqueous OAF solutions. The main features of the OAF solution behavior are summarized in Scheme 1. (b) Spectroscopic Properties of Surfactant Micelle-Sorbed Probes. UV-vis Absorption Spectra. In order to understand spectra of probes sorbed in the core/ shell interfacial region of PS-PMA micelles, the spectral properties of probes bound at sodium dodecyl sulfate (SDS) anionic micelles were investigated firstly. SDS is one of the most widely studied surfactants, and reliable micellization data are available in the literature.13 Despite the fact that both DAF and OAF are negatively charged at (13) (a) Phillips, J. N. Trans. Faraday Soc. 1955, 51, 561. (b) Ford, W. P. J.; Ottewill, R. H.; Parreira, H. C. J. Colloid Interface Sci. 1966, 21, 522. (c) Kratohvil, J. J. Colloid Interface Sci. 1980, 75, 271.
Polyelectrolyte Behavior of the PMA Shell
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Scheme 1. Dissociation and Association Processes in Aqueous OAF Solutions (depending on OAF concentration, cF, pH, and ionic strength of the solution, I) That Affect the Fluorescent Behavior of OAF
Figure 6. Normalized fluorescence intensity, IF/I max F , vs pH, of 3 µM DAF and OAF in pure buffer solution (curves 1 and 4, respectively), in the presence of SDS micelles (curves 2 and 5, respectively) and in the presence of PS-PMA micelles (curves 3 and 6, respectively). SDS and PS-PMA concentrations were 10 mmol‚L-1 and 0.12 g‚L-1, respectively.
Figure 5. Absorption spectra of 3 µM DAF in the presence of PS-PMA micelles at various pH values. Insert: Absorption spectra of 3 µM DAF in the presence of SDS micelles at various pH values. SDS and PS-PMA concentrations were 10 mmol‚L-1 and 0.12 g‚L-1, respectively.
high pH, they bind very strongly to SDS micelles. Absorption spectra of DAF sorbed onto SDS micelles are shown in Figure 5, insert. All absorption maxima are redshifted as compared with the free DAF in the solution (for the dianion, the shift is ca. 6 nm). A careful comparison with the spectra of free DAF reveals that the extinction coefficient of the neutral dye is enhanced after binding to the SDS micelle and the onset of the dianion absorption is shifted to higher pHs as compared with the free DAF in the solution. The latter effect is consistent with conclusions from the Fromherz theory14 and will be discussed later in detail. The absorption spectrum of the bound OAF (not shown) is very similar to that of the bound DAF, which indicates the similar interactions of the fluorescein head-groups with the microenvironment in both cases. Fluorescence Measurements. Normalized fluorescence intensity as a function of pH for both SDS micelle-sorbed probes is shown in Figure 6. All curves are normalized by the corresponding maximum intensities obtained in highly alkaline solutions. Curves 2 and 5 depict dependencies on pH for the sorbed DAF and OAF, respectively. Curves for nonsorbed probes in corresponding solutions are also shown for comparison: dashed curves 1 and 4 for free DAF and OAF, respectively. It should be pointed out that the absolute intensities of the free and the micelle-sorbed (14) Ferna´ndez, M. S.; Fromherz, P. J. Phys. Chem. 1977, 81, 1755.
DAF are comparable, while the OAF intensity increases ca. 10 times after binding to SDS micelles. The significant difference between absorption and emission spectra of the free and the micelle sorbed DAF is consistent with the classical Ferna´ndez and Fromherz theory.14 The observed shift in the position of the sigmoidal part of the curve in the pH scale corresponds to the decreased acidity of the fluorescein head-group after the sorption of the probe at the interface. It may be accounted for (i) by a lower polarity of the microenvironment and (ii) by additional Gibbs energy that is necessary for dissociation of -COOH close to the negatively charged surface. The extra-free-energy arises from the electrostatic repulsion that accompanies the formation of the bound anion in the vicinity of a number of bound -COO- anions and the proton escape across the barrier formed by positively charged counterions, i.e., across the electric bilayer, into the bulk solvent. Both effects hinder the dissociation of phenolic groups of the fluorescein head group of the micelle-sorbed DAF. As concerns the comparison of corresponding curves for DAF and OAF, the differences may be easily understood. Positions of the sigmoidal parts in curves 2 and 5 in the pH scale are similar, which indicates similar interactions of fluorescent head-groups for both SDS micelle bound probes. This finding agrees with conclusions from absorption measurements. Significant differences between curves 1 and 4 for the free DAF and OAF, respectively, have been already discussed (see the discussion concerning Figure 4). The shift of the sigmoidal part of the curve for the bound OAF to the lower pH, contrary to the theoretical expectation, is consistent with the observation that the pH-dependent fluorescence of free OAF is strongly influenced by hydrophobic association of OAF in the solution, which results in the fluorescence self-quenching. The emission at 520 nm does not therefore reflect the degree of dissociation of phenolic groups in the solution. The comparison of fluorescence intensities vs pH for DAF sorbed at the interface of the SDS and the Triton micelles allows evaluation of the surface potential of SDS micelles. The measurement leads to values that are consistent with that reported by Fromherz. Since the probes bound to SDS micelles were studied as the reference system only, we neither show nor discuss the known conclusions.
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(c) Polyelectrolyte Behavior of PMA Micellar Shells and Fluorescence of Probes Bound at the Core-Shell Interface of Polyelectrolyte Micelles. The polyelectrolyte shell of the multimolecular PS-PMA micelle represents a very specific polyelectrolyte assembly in aqueous media that does not behave as a usual polyelectrolyte solution. Since the polystyrene cores of polymeric micelles are kinetically frozen in water, the shell-forming polyelectrolyte blocks may be regarded as weak polyelectrolyte brushes formed by fairly long chains that are tethered to a significantly curved surface. Individual chains are partially oriented in the radial direction. Density of polymer segments is very high close to the core/ shell interface and decreases continuously toward the shell periphery. A number of theories of polyelectrolyte brushes have been published recently.15 Papers by Misra and Mattice15a or papers by Zhulina, Fleer, et al.15e describe basic properties of polymeric brushes formed by strong, as well as weak, polyelectrolyte, e.g., conformational changes of the brush-forming chains with changing pH and ionic strength reasonably well. Poly(methacrylic acid) is one of the most studied polyelectrolytes due, in part, to its practical importance and to its interesting properties. A number of experimental works show that linear PMA does not behave as a typical weak polyelectrolyte.16 It undergoes strong hypercoiling with decreasing pH in the region of pH 6-5. Since the micellar shell is formed by PMA, it represents an unusual polyelectrolyte brush and its behavior is very complex. Indirect fluorometric measurements indicate that the inner nonpolar layer of the shell resembles the hydrophobic domains in hypercoiled linear PMA, except that the former is more dense and more hydrophobic.1,5d,e We have shown recently that the correct interpretation of the pH-dependent shell behavior requires taking into account also the Donnan equilibria between shells and the bulk solution.17 UV-vis Absorption and Fluorescence Measurements. In order to explain the strategy of our fluorescence measurements with the tagged micelles and the amphiphilic fluorophores and the goals we follow, the most important features of the behavior of the studied system are depicted in Scheme 2. It shows the structure and behavior of micelles and the association and binding of amphiphilic fluorophores in acid and basic aqueous solutions. Further it shows schematically the degree of dissociation of carboxylic groups within PMA shells as a function of the distance from the core/shell interface at (15) (a) Misra, S.; Mattice, W. L. Macromolecules 1994, 27, 2058. (b) Mattice, W. L. In Polymer and Solvent Organization; Webber, S. E., Munk, P., Tuzar, Z., Eds.; NATO ASI, 1995. (c) Seidel, Chr. Macromolecules 1994, 27, 7085. (d) Israe¨ls, R.; Leermakers, F. A. M.; Fleer, G. L. Macromolecules 1994, 27, 3087. (e) Lyatskaya, Yu. V.; Leermakers, F. A. M.; Fleer, G. J.; Zhulina, E. B.; Birshtein, T. M. Macromolecules 1995, 28, 3562. (f) Zhulina, E. In Polymer and Solvent Organization; Webber, S. E., Munk, P., Tuzar, Z., Eds.; NATO ASI, 1995. (16) (a) Katchalski, A. J. Polymer Sci. 1951, 7, 393. (b) Arnold, R. J. Colloid Sci. 1957, 1, 549. (c) Anufrieva, E. V.; Birshtein, T. M.; Nekrasova, T. N.; Ptitsyn, C. B.; Scheveleva, T. V. J. Polym. Sci., Part C 1968, 16, 3519. (d) Delben, F.; Crezcenzi, V.; Quadrifoglio, F. Eur. Polym. J. 1972, 8, 933. (e) Koenig, J. L.; Angood, A. C.; Semen, J.; Lando, J. B. J. Am. Chem. Soc. 1969, 91, 7250. (f) Ghiggino, K. P.; Tan, K. L. In Polymer Photophysics; Phillips, D., Ed.; Chapman and Hall: London, 1985; Chapter 7. (g) Tan, K. L.; Treolar, F. E. Chem. Phys. Lett. 1980, 73, 239. (h) Bedna´rˇ, B.; Morawetz, H.; Shafer, J. A. Macromolecules 1985, 18, 1940. (i) Wang, Y.; Morawetz, H. Macromolecules 1986, 19, 1925. (j) Bedna´rˇ, B.; Trneˇna´, J.; Svoboda, P.; Vajda, Sˇ .; Fidler, V.; Procha´zka, K. Macromolecules 1991, 24, 2054. (17) (a) Munk, P.; Tuzar, Z.; Procha´zka, K. Collect. Czech. Chem. Commun. 1997, 62, 1730. (b) Karymov, M. A.; Procha´zka, K.; Mendenhall, J. M.; Martin, T. J.; Munk, P.; Webber, S. E. Langmuir 1996, 12, 4749.
S ˇ teˇ pa´ nek et al. Scheme 2. An Outline of Various Processes in Aqueous Mixtures of OAF with PS-PMA Micellesa
a(a) The expansion of PMA shells with pH, leading to the increase in the hydrodynamic radius, RH (schematically according to ref 17). (b) Degree of dissociation of -COOH group in PMA shells as a function of the distance from the core/shell interface and the effect of pH on PMA dissociation (schematically according to ref 15e). (c) Effect of pH on the structure of PSPMA micelles and binding of OAF in the core/shell interfacial region with the aliphatic tail partially buried in the PS core and the fluorescein head-group trapped in the inner hydrophobic PMA layer.
different pH15e and changes in micellar size with pH17b that have to be taken into account in the discussion of experimental results. At first, we wanted to assess how strongly the probes are bound to micelles. Since both DAF and OAF contain aliphatic chains of the length ca. 2 and 3 nm, respectively, that interact with the nonpolar core and secure the sorption of the probe onto the polymeric micelle, we can assume that the fluorescein head-groups are preferentially trapped in the inner hydrophobic part of the shell. Information on the partition equilibrium of probes between micellar shells and the aqueous phase was obtained by nonradiative energy transfer measurements in fluorescently tagged PS-PMA micelles with an energy donor covalently bound between the PS and the PMA block and OAF (or DAF) sorbed at the interface. Molar mass, composition, and micellization properties of the fluorescent sample, that is tagged by a pendant 9,10-diphenylanthracene (DPA) group between the PS and PMA blocks (on average 1.8 tags per chain), are very similar to those of the nontagged copolymer.1,6 In PS-PMA micelles, all DPA tags are located very close to the core/shell interface. They prefer the nonpolar environment and are mostly trapped inside the coresat its outermost periphery. Due, in part, to steric and to thermodynamic reasons (finite chain flexibility, optimum enthalpy-to-entropy balance of the system which includes also the minimization of the interfacial tension), the PS and PMA chains are partially intermixed in the interfacial region and also some DPA groups are displaced outside the core into the hydrophobic PMA layer. We have shown recently that a relatively small fraction of DPA fluorescence may be quenched by watersoluble quenchers (e.g., by thallium salts).6 Since the thallium quenching assumes the collision mechanism, a non-neglible, but small, fraction of DPA tags must be accessible for Tl+ cations.
Polyelectrolyte Behavior of the PMA Shell
Figure 7. Ratio of the OAF-to-DPA fluorescence intensities, I OAF /I DPA , of OAF, sorbed to PS-DPA-PMA micelles, vs micelle F F concentration, cp. Insert: Normalized absorption and emission spectrum of DPA (curves 1 and 2, respectively), together with absorption and emission spectrum of OAF (curves 3 and 4, respectively).
The emission band of DPA overlaps strongly with the absorption band of OAF (see the insert in Figure 7), and therefore these two fluorophores represent a good pair for nonradiative energy transfer studies. In our older study, we have estimated the Fo¨rster radius, R0 ca. 4.6 nm, for the alkaline OAF environment.1a The large value of R0 is mainly the result of a large value of the OAF extinction coefficient. The Fo¨rster radius is pH-dependent and decreases with decreasing pH. Even though the large R0 value precludes the direct proof that the fluorescein headgroup of OAF is localized in the immediate vicinity of the interface, very strong quenching of the DPA fluorescence and strong OAF emission allows for a rough estimate of the partition coefficient. The estimate was obtained as follows. A volume of 100 µL of the 10-6 M solution of OAF in a borate buffer, pH 9, was added to 3 mL of the micellar solution (cP ) 3.5 × 10-3 g/mL), and the stock mixture was left overnight to equilibrate. A series of solutions with decreasing micelle concentration (however with a constant probe-to-micelle molar ratio) was prepared by mixing the stock solution with the buffer and left to equilibrate. The energy donor (DPA) was excited at 359 nm, and both the DPA fluorescence intensity, IDPA at 411 nm, and the OAF fluorescence intensity, IOAF at 520 nm, were measured. With increasing dilution, relative concentration of OAF in the bulk buffer increases and concentration of the bound OAF decreases. As a consequence, the effect of the nonradiative energy transfer (NRET) on the fluorescence spectra decreases. Ratio of the acceptor-to-donor intensities, IFOAF/IFDPA, as a function of log cP is shown in Figure 7. The measured ratio is not directly proportional to the ratio of concentrations of individual OAF forms; however the shape of the curve with two slightly ascending parts and a sudden sigmoidal increase in between allows the order of magnitude of the partition coefficient in alkaline solution, Kf, 105. If we take into account the non-negligible solubility of OAF in alkaline solutions, the large value Kf shows very strong affinity of OAF to the studied micelles. The assumption that the fluorescein head-group of the micelle-bound OAF is trapped and immobilized in the compact and hydrophobic environment in the inner part of the shell is supported also by fluorescence anisotropy measurements. Steady-state fluorescence anisotropy, 〈r〉, of the directly excited micelle-bound OAF (excitation at 490 nm, emission at 520 nm) is fairly high even in alkaline
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solutions (it decreases slightly with increasing pH, e.g., 〈r〉 ) 0.33 for pH 5 and 0.27 for pH 9). Since the fluorescence lifetimes are almost constant in this pH region, τF ca. 4.5 ns, the steady-state anisotropy is a good indicator of the probe mobility. Increasing pH promotes the dissociation of carboxylic groups in the shell. In the region of neutral to slightly alkaline pHs, it concerns mainly the middle part of the shell and, in alkaline buffers, also the inner part of the shell is affected. The innermost part close to the core is influenced only little. Nevertheless the strong electrostatic repulsion of many dissociated carboxylic groups, that is not fully compensated by counterions, causes a significant stretching of the shell (mainly the stretching of the outer part)15d,17b and a certain pull of PMA blocks in the radial direction toward the bulk. It results in a moderate decrease in the inner shell compactness. The mobility of probes trapped in the inner layer of the shell increases with rising pH; however, the anisotropy measurements show that the microviscosity of the medium is fairly high in the whole pH region. UV-vis absorption spectra of DAF and OAF bound to PS-PMA micelles were measured in a broad range of pH and ionic strength to get information on the interaction of the fluorescein head-group with the PMA shell. The DAF and OAF spectra for given pH are almost identical and do not depend on ionic strength. Spectra for DAF are shown in Figure 5. Position of the absorption bands and the general trends are similar to those for the SDS micellebound probes (see Figure 5, insert), except that the absorption band for the neutral form of the dye is less developed and a non-negligent dianion absorption is observed even in the region of very low pHs (close to pH 2). The comparison suggests that the micropolarity of the interface in SDS and in PS-PMA micelles are similar; however, any conclusions may be drawn only with the maximum care and precaution. As concerns the SDS micelles, there is a distinct electric bilayer at the interface, while properties of the shell in PS-PMA micelles change relatively smoothly within a relatively long distance. Normalized fluorescence intensities of PS-PMA-bound probes are shown in Figure 6, curves 3 and 6, for DAF and OAF, respectively. The sigmoidal increase in (IF/Fmax) vs pH for both probes occurs approximately in the same pH region as that for the SDS micelle-bound probes. The curve for the PS-PMA-bound OAF differs from all other curves for the SDS and PS-PMA micelles-bound probes. The slope is less steep and the curve does not drop to zero at low pH. So far, we do not fully understand the reason for the relatively strong fluorescence of the PS-PMA micellebound OAF in acid solutions. Supplementary studies are in progress, and the results will be reported in future. From the experimental point of view, the significant fluorescence at low pH is advantageous since it allows for studying the OAF binding to polymeric micelles in acid solutions. Polyelectrolyte PS-PMA micelles are in a kinetically frozen state in aqueous media which means that, in contrast to SDS micelles, very dilute solutions of PS-PMA micelles may be prepared in aqueous buffers without any danger of micelle dissociation or reorganization.1,5 While in spectroscopic studies of OAF (or DAF)-SDS systems, the ratio of OAF-to-SDS was ca. 1/1, we were able to cover a much broader range of relative concentrations in measurements with polymer micelles. We were interested mainly in systems containing high numbers of probes per single polymeric micelle. With increasing number of probes per micelle, the average distance between individual sorbed probes decreases and the probability of the probeprobe interactions and formation of probe aggregates
4192 Langmuir, Vol. 15, No. 12, 1999
S ˇ teˇ pa´ nek et al.
Figure 9. S1-absorption maximum, λmax (curve 1), and mean fluorescence lifetime, 〈τ〉 (curve 2), of DAF in the presence of PS-PMA micelles, as a function of the DAF-to-micelle molar ratio, ξ.
Figure 8. (a) Absorption spectra, (b) fluorescence spectra (exc. 490 nm), and (c) fluorescence decays (exc. 490 nm, em. 520 nm) of 3 µM DAF in the presence of PS-PMA micelles. Insert in (b): The fluorescence spectra normalized by the maximum intensity. PS-PMA concentration was 0.01 g‚L-1 (curve 1), 0.12 g‚L-1 (curve 2), and 1.20 g‚L-1 (curve 3), pH 9.2.
increases. UV-vis absorption spectra of DAF bound to PS-PMA micelles were measured for a series of alkaline solutions (pH 9.2) differing in DAF-to-micelle molar ratio, ξ. Selected spectra (normalized for a constant dye concentration) are shown in Figure 8a. For low ξ (curve 1), individual fluorescein head-groups of the sorbed DAF are trapped in the inner hydrophobic PMA layer far from each other. The red shift (ca. 8 nm) indicates that the probes experience relatively nonpolar environment and do not
form H-dimers. In the range ξ from 20 to 500, the spectra change (cf. curve 2 in Figure 8a). The maximum in the absorption band shifts to lower wavelengths and the a transient broadening of the absorption band is observed. Concentration of the fluorescein head-groups at the interface increases, and some probes form H-aggregates. At high ξ, the absorption band is blue-shifted and narrower than that for intermediate ξ, indicating thus formation of H-aggregates. Position of the absorption maximum as a function of ξ is shown in Figure 9. If we assume that the sorption equilibrium is shifted in favor of the sorbed probe (which is justified on the basis of our NRET measurements), it is possible to calculate the average distance between fluorescein head-groups for a given ξ. Radius of the core of the studied micelles measured by small-angle neutron scattering18 is ca. 11 nm, which means that the surface area per one sorbed probe head-group is ca. 7.0 nm2 for a DAF-to-micelle ratio, ξ ) 200. Since the flat fluorescein molecule is fairly large, the further increase in the number of the absorbed probes requires some sort of head-group orientation which leads to the massive formation of H-aggregates at the core/shell interface. Interesting supplementary information may be obtained by the steady-state and time-resolved fluorometry. Steadystate spectra are shown in Figure 8b for the same systems as in Figure 8a, and the corresponding time-resolved fluorescence decays are shown in Figure 8c. To secure the comparable and sufficient fluorescence intensity and thus the reasonable accuracy of all measurements, the DAF concentration was kept constant in all solutions, while that of PS-PMA varied. Correct interpretation of the data requires taking into account the shifts in the sorption equilibria due (i) to the changes in the DAF-to-micelle molar ratio and (ii) to the changes in the micelle concentration in the solution (i.e., to changes in micelleto-solvent molar ratio). Conclusions that may be drawn from fluorescence measurements (from the maxima positions, absolute intensities and from the fluorescence lifetimes) are consistent with those based on the absorption measurements and may be summarized as follows (see Figure 9). (i) At low DAF-to-micelle molar ratio, ξ (i.e., at high micellar concentrations), almost all probes are sorbed in the nonpolar and fairly viscous micellar interfacial region and their concentration in the bulk solution is negligible. Fluorescence maximum is close to 522 nm, mean fluorescence lifetime is ca. 4.6 ns, and the fluores(18) Plesˇtil, J.; Krˇ´ızˇ, J.; Procha´zka, K.; Webber, S. E.; Munk, P.; Wignall, G. Macromolecules, in press.
Polyelectrolyte Behavior of the PMA Shell
cence quenching is suppressed. The H-associates do not form under these conditions and all the other quenching mechanisms are also suppressed in the highly viscous medium. (ii) As the micelle concentration decreases (with the DAF concentration remaining constant), ratio ξ increases and the interfacial region approaches the saturation by the fluorescein head-groups. Formation of H-associates leads to a significant fluorescence quenching which is manifested by a considerable decrease in the steady-state fluorescence intensity and fluorescence lifetimessee curve 2 in Figure 8b and curve 2 in Figure 9, respectively. As concerns the fluorescence lifetimes, the micelle-bound dyes are partially protected against the oxygen quenching in the compact inner layer of the PMA shell and the fluorescence lifetime (ca. 4.6 ns) is slightly higher than that of the dye in the solution (ca. 4.3 ns). However, an apparent dynamic self-quenching of the bound DAF fluorescence was observed for the probe-tomicelle molar ratios higher than 20. Under these conditions, a two-component decay of the fluorescence appears with one lifetime ca. 0.6-1.0 ns, and the second ca. 3.84.3 ns. We assume that the fast component is from the fairly closely packed DAF head-groups in the innermost part of the shell, where the self-quenching may occur, and the slow component corresponds to the fluorescence from the probes in the outer part of the shell and in the bulk solution. For ξ higher than 200 and relatively high micelle concentrations, the latter contribution dominates and the decay is single-exponential. The mean fluorescence lifetime,19 which is the average value from various decays of individual probes that interact with their microenvironments, drops to ca. 3 ns in the minimum region for ξ ca. 100-200. Under these conditions, some fluorescein head-groups are probably displaced from the innermost part of the shell into more polar environment and their emission is slightly redshifted as compared with low ξ solutions. (iii) At very high ξ, the core/shell interface is saturated by DAF and the fluorescence of the bound DAF is strongly quenched. (19) The measured fluorescence decays were fitted by the twoexponential function, IF(t) ) R1 exp(-t/τ1) + R2 exp(-t/τ2). It provided a good fit in all cases, with χ2 less than 1.1. The mean fluorescence lifetime, 〈τ〉, was calculated from the formula 〈τ〉 ) (R1τ12 + R2τ22)/(R1τ1 + R2τ2).
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However, the ratio of the micelle-bound-to-dissolved DAF is shifted in favor of the dissolved DAF due to very low micelle concentration and a fairly strong emission with the lifetime ca. 4.3 ns is observed from the water-dissolved DAF. Both the fluorescence intensity and the mean fluorescence lifetime, 〈r〉 vs ξ show well pronounced minima for ξ ca. 100-200. Conclusions 1. Both the S1-absorption band (close to 500 nm) and the fluorescence band (around 522 nm) of the PS-PMA micelle-bound probes (DAF and OAF) are red-shifted in comparison with the free probes (492 and 514 nm, respectively). The red shift is caused by weaker hydrogen bond interactions between the probe and its microenvironment in the inner part of the PMA shell than in the aqueous phase. 2. Significant differences in the absorption spectra of the fluorescein, DAF, and OAF in aqueous solutions show that OAF forms associates in a broad pH region, while DAF associates only at low pH. 3. Probes sorbed onto polymeric micelles at low probeto-micelle molar ratio, ξ, are relatively far from each other and do not form H-aggregates. With increasing ξ, the distances between probes decrease which results in a massive formation of H-aggregates. 4. Apparent pKa describing the -OH dissociation of the micelle-bound DAF, determined from fluorescence measurements, is higher than that of the free dye. This observation is consistent with the classical Fromherz theory. Fluorescent behavior of OAF is more complex due to the association of the dye in aqueous media. The unexpected pKa shift upon OAF binding to PS-PMA micelles may be understood qualitatively if the interplay of several competing effects is taken into account. Acknowledgment. K.P. is grateful for the financial support by the Grant Agency of the Czech Republic (Grant 97/203/0249) and by the Charles University in Prague (188/97/B-Ch/PrF). S.E.W. is grateful for the support by the U.S. Army Office of Research (Grant DAAH04-95-1017) and the Robert A. Welch Foundation (F-356). LA981129D
