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Interaction of the Tetra(4-sulfonatophenyl)porphyrin with Ionic Surfactants: Aggregation and Location in Micelles Shirley C. M. Gandini, Victor E. Yushmanov, Iouri E. Borissevitch,† and Marcel Tabak* Instituto de Quı´mica de Sa˜ o Carlos, Universidade de Sa˜ o Paulo, C.P. 780, 13560-970 Sa˜ o Carlos, SP, Brazil Received February 2, 1999. In Final Form: May 14, 1999 Interactions of the water-soluble tetra(4-sulfonatophenyl)porphyrin (TPPS4) with ionic micelles in aqueous solutions have been studied by optical absorption, fluorescence, resonance light-scattering (RLS), and 1H NMR spectroscopies. The presence of three different species of TPPS4 in cationic cetyltrimethylammonium chloride (CTAC) solution has been unequivocally demonstrated: free porphyrin monomers, monomers bound to micelles, and nonmicellar porphyrin/surfactant aggregates. This result is similar to our previous findings for TPPS4 interactions with biomacromolecules (serum albumin and DNA). The surfactant:porphyrin ratio for maximum aggregate formation is around 4:1-5:1 and 14:1 at pH 3.0 and pH 7.5, respectively. In the case of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) the aggregates were not observed. Binding constants estimated from these data were of the order of 104 M-1 for CTAC and HPS. Our data show that solubilization of porphyrins within nonpolar regions of micelles is determined, in general, by nonspecific hydrophobic interactions, yet it is significantly modulated by electrostatic factors. NMR chemical shift, T1, and nuclear Overhauser effect data indicate that TPPS4 is located mainly in the hydrophobic core in CTAC and HPS micelles, while in lysophosphatidylcholine its involvement in the polar area is more significant.
Introduction The importance of porphyrins and related compounds as therapeutic drugs and targeting agents has been widely recognized.1,2 High affinity and phototoxicity of sulfonated meso-tetraphenylporphyrins to tumors make them promising compounds for photodynamic therapy (PDT)3 and tumor localization (via fluorescence endoscopy). Metal derivatives of anionic tetra(4-sulfonatophenyl)porphyrin (TPPS4) have been considered as prototypes for tumorspecific contrast agents in radiological and magnetic resonance imaging.4,5 The mechanisms of biological effects of porphyrins and metalloporphyrins may involve their penetration through membranes6 and binding to proteins, e.g., to albumins in blood plasma,7 while the porphyrin aggregation may decrease their activity as sensitizers8 and contrast agents.9 Using 1H NMR, ESR, and optical spectroscopic techniques, we have reported elsewhere the solution properties10 and aggregation of free bases TPPS4 * To whom correspondence should be addressed. Tel.: ++55 (16) 273-9979. Fax: ++55 (16) 273-9976. E-mail:
[email protected]. † On leave from the Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia. Present address: Departamento de Fı´sica, Universidade Federal de Pernambuco, Recife, PE, Brazil. (1) Bonnett, R. Chem. Soc. Rev. 1995, 24, 19-33. (2) Cannon, J. B. J. Pharm. Res. 1993, 82, 435-446. (3) Berg, K.; Bommer, J. C.; Winkelman, J. W.; Moan, J. Photochem. Photobiol. 1990, 52, 775-781. (4) Hambright, P.; Fawwaz, R.; Valk, P.; McRae, J.; Bearden, A. J. Bioinorg. Chem. 1975, 5, 87-92. (5) Nelson J. A.; Schmiedl U. Magn. Reson. Med. 1991, 22, 366-371. (6) Ricchelli, F.; Gobbo, S.; Jori, G.; Moreno, G.; Vinzens, F.; Salet, C. Photochem. Photobiol. 1993, 58, 53-58. (7) Datta-Gupta, N.; Malakar, D.; Dozier, J. Res. Commun. Chem. Pathol. Pharmacol. 1989, 63, 289-292. (8) Keene, J. P.; Kessel, D.; Land, E. J.; Redmond, R. W.; Truscott, T. G. Photochem. Photobiol. 1986, 43, 117-120. (9) Lyon, R. C.; Faustino, P. J.; Cohen, J. S.; Katz, A.; Mornex, F.; Colcher, D.; Baglin, C.; Koenig, S. H.; Hambright, P. Magn. Reson. Med. 1987, 4, 24-33. (10) Yushmanov, V. E.; Imasato, H.; Tominaga, T. T.; Tabak, M. J. Inorg. Biochem. 1996, 61, 233-250.
and tetra(N-methyl-4-pyridyl)porphyrin (TMPyP) and their Fe(III) and Mn(III) complexes upon their binding to bovine serum albumin (BSA)11-14 and DNA.15-17 Our results suggested that the features of aggregation of different water-soluble meso-tetraaryl-substituted porphyrins on various biological structures have much in common. In this study, an attempt is made to gain more insight into the nature of TPPS4 interaction with biological structures using the simplest models for membranes and protein reaction centers: aqueous ionic micelles18,19 with different headgroup charge. Free base porphyrins, besides being potential PDT drugs, also serve to some extent as diamagnetic analogues for paramagnetic metallocomplexes. Although the paramagnetism may be advantageous in NMR studies,20,21 this approach is limited because of the strong displacement and broadening of NMR peaks and fluorescence quenching. The fact that some dyes show the same color changes in the presence of highly charged colloids as they do in the presence of biological materials22 justifies the use of micelle models to study the porphyrin (11) Yushmanov, V. E.; Tominaga, T. T.; Borissevitch, I. E.; Imasato, H.; Tabak, M. Magn. Reson. Imaging 1996, 14, 255-261. (12) Borissevitch, I. E.; Tominaga, T. T.; Imasato, H.; Tabak, M. J. Lumin. 1996, 69, 65-76. (13) Tominaga, T. T.; Yushmanov, V. E.; Borissevitch, I. E.; Imasato, H.; Tabak, M. J. Inorg. Biochem. 1997, 65, 235-244. (14) Borissevitch, I. E.; Tominaga, T. T.; Imasato, H.; Tabak, M. Anal. Chim. Acta 1997, 343, 281-286. (15) Gandini, S. C. M.; Borissevitch, I. E.; Perussi, J. R.; Imasato, H.; Tabak, M. J. Lumin. 1998, 78, 53-61. (16) Borissevitch, I. E.; Gandini, S. C. M. J. Photochem. Photobiol. 1998, B43, 112-120. (17) Gandini, S. C. M.; Yushmanov, V. E.; Perussi, J. R.; Tabak, M.; Borissevitch, I. E. J. Inorg. Biochem. 1999, 73, 35-40. (18) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-Interscience: New York, 1982. (19) Yushmanov, V. E.; Imasato, H.; Perussi, J. R.; Tabak, M. J. Magn. Reson. B 1995, 106, 236-244. (20) Yushmanov, V. E. Inorg. Chem. 1999, 38, 1713-1718. (21) Mazumdar, S. J. Phys. Chem. 1990, 94, 5947-5953. (22) Deumie´, M.; El Baraka, M. J. Photochem. Photobiol. 1993, A74, 255-266.
10.1021/la990108w CCC: $18.00 © 1999 American Chemical Society Published on Web 07/16/1999
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binding to natural cell components. Various synthetic and natural porphyrins incorporated into micelles and lipid bilayers have been extensively used for biomimetic purposes.23-28 It has been suggested that hydrophobic porphyrins can penetrate the lipid regions of the membranes and distribute into protein-rich membrane domains,6 while highly polar species were supposed to partition mainly in the aqueous compartments.29 However, the interaction of water-soluble synthetic porphyrins with ionic micelles has been clearly shown.30,31 In the presence of ionic surfactants below their critical micelle concentration (cmc), both TPPS432-34 and some meso-tetraaryl-substituted picket fence porphyrins35 were shown to form aggregates. Above cmc, micelles are usually considered only as a means to solubilize the aggregates of porphyrin derivatives into monomers.27,30-32,36 However, aggregation of tetraphenylporphyrin derivatives (sulfonated or not) in micelles and bilayers formed by surfactants and short peptides has also been reported,20,28,37,38 in contrast to protoporphyrin and its derivatives.27,28,36 Thus, studies of interaction of water-soluble porphyrin derivatives with biomimetic systems are relevant in view of the challenges faced in formulating these drugs into stable, effective, and safe-dosage forms. Studies of the micellar systems may also shed more light on the interaction of ionic porphyrins with liposomes used for porphyrin drug delivery. Materials and Methods Materials. Sodium salt of TPPS4 and TMPyP chloride (Midcentury), lysophosphatidylcholine (LPC, Sigma), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, Sigma), sodium dodecyl sulfate (SDS, BioRad), polyoxyethylene lauryl ether (Brij-35, Sigma), D2O (Merck), and sodium acetate and potassium phosphate (Mallinckrodt) were used as purchased. Cetyltrimethylammonium chloride (CTAC, Herga) was purified by acetone-methanol extraction. The structures, micellar parameters of the porphyrin and surfactants, and details of sample preparation are presented in the Supporting Information. Optical Absorption, Fluorescence, and Light Scattering Experiments. (a) Techniques. Optical absorption spectra were measured on a Cary 5G UV-vis-NIR spec(23) Tsuchida, E.; Nishide, H. Top. Curr. Chem. 1986, 132, 63-100. (24) van Esch, J.; Roks, M. F. M.; Nolte, R. J. M. J. Am. Chem. Soc. 1986, 108, 6093-6094. (25) Groves, J. T.; Neumann, R. J. Am. Chem. Soc. 1989, 111, 29002909. (26) Robinson, J. N.; Cole-Hamilton, D. J. Chem. Soc. Rev. 1991, 20, 49-94. (27) Mazumdar, S.; Medhi, O. K.; Mitra, S. Inorg. Chem. 1988, 27, 2541-2543. (28) Maiti, N. C.; Mazumdar, S.; Periasamy, N. J. Phys. Chem. 1995, 99, 10708-10715. (29) Ricchelli, F.; Jori, G. Photochem. Photobiol. 1986, 44, 151-157. (30) Kadish, K. M.; Maiya, G. B.; Araullo, C.; Guilard, R. Inorg. Chem. 1989, 28, 2725-2731. (31) Kadish, K. M.; Maiya, G. B.; Araullo-McAdams, C. J. Phys. Chem. 1991, 95, 427-431. (32) Maiti, N. C.; Mazumdar, S.; Periasamy, N. Curr. Sci. 1996, 70, 997-999. (33) Tominaga, T.; Endoh, S.; Ishimaru, H. Bull. Chem. Soc. Jpn. 1991, 64, 942-948. (34) Schmehl, R. H.; Whitten, D. G. J. Phys. Chem. 1981, 85, 34733480. (35) Barber, D. C.; Freitag-Beeston, R. A.; Whitten, D. G. J. Phys. Chem. 1991, 95, 4074-4086. (36) Simplicio, J. Biochemistry 1972, 11, 2525-2529. (37) van Esch, J. H.; Feiters, M. C.; Peters, A. M.; Nolte, R. J. M. J. Phys. Chem. 1994, 98, 5541-5551. (38) Venkatesh, B.; Jayakumar, R.; Pandian, R. P.; Manoharan, P. T. Biochem. Biophys. Res. Commun. 1996, 223, 390-396.
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trophotometer monitoring either the whole spectrum or the Soret band and Q bands separately. The extinction coefficients at the maximum Soret band for TPPS4, used to determine concentrations, were 3.92 × 105 M-1 cm-1 at pH 3.0 and 5.42 × 105 M-1 cm-1 at pH 7.5.12 Fluorescence and light-scattering spectra were recorded on a Jasco FP777 spectrofluorimeter. To measure the fluorescence changes induced by the addition of surfactants, excitation at λex ) 575 nm and λex ) 525 nm at pH 7.5 and pH 3.0 was used, respectively. The integral fluorescence intensity was monitored in the range from 600 to 750 nm. The scattered light intensity was monitored using the rightangle geometry in the synchronous scanning mode of excitation and emission monochromators in the range from 350 to 600 nm. The experimental light-scattering spectra were corrected, taking into account the solution optical absorption and the instrument-sensitivity dependence on the wavelength as described elsewhere.14 Experiments were performed at room temperature (25 ( 2) °C. All experimental data are the averaged values of at least three independent experiments. The absorption and emission spectra obtained in the pH or surfactant concentration titrations were analyzed by the convex constraint algorithm (CCA)39 (a software kindly provided by Dr. G. D. Fasman) to determine the contributions of individual spectral components. (b) pKa and Binding Constant Determinations. The pKa values of the porphyrin in the presence or absence of surfactants were determined by pH titrations using optical absorption and fitting the data to the titration equation for the protonation equilibrium for the central nitrogens Ka
(H2TPPS4)4- + 2H+ 798 (H+)2(H2TPPS4)4-
(1)
as described previously40 and given in detail in the Supporting Information. The titration of TPPS4 in solution as a function of the concentration of different surfactants allows the evaluation of the association constants of TPPS4 with the surfactants, Kb. The optical absorption data were fit to the equation for the binding equilibrium as described previously40 and given in detail in the Supporting Information. NMR Experiments. Proton NMR spectra were run on a Bruker AC-200 spectrometer (resonant frequency, 200.13 MHz) at 23 °C. The residual water peak was presaturated by gated irradiation during the preacquisition delay of 2 s. The 11° pulses of 1-µs duration were repeated every 4.8 s, the bandwidth was 2940 Hz, and the memory size was 16 K data points. The postprocessing exponential filter with a line-broadening factor of 0.2 Hz was applied in the time domain. Chemical shifts were referenced to 2,2dimethyl-2-silapentane-5-sulfonate (DSS, in D2O). Nonselective relaxation times T1 were measured with the inversion-recovery pulse sequence (t-π-τ-π/2acquire) with a π/2 pulse duration of 8.2 µs. Selective T1 relaxation times were measured with the same pulse scheme, where the π pulse was given by the proton decoupler gated at the chosen frequency for 36 ms. The residual water resonance was irradiated except during the pulses and acquisition. Truncated NOE-difference experiments in the presence of micelles were performed by using a repetition time of 5.5 s and a preirradiation time of 50-400 ms. Typically, 160 scans were accumulated for each irradiation fre(39) Perczel, A.; Park, K.; Fasman, G. D. Anal. Biochem. 1992, 203, 83-93. (40) Perussi, J. R.; Yushmanov, V. E.; Monte, S. C.; Imasato, H.; Tabak, M. Physiol. Chem. Phys. Med. NMR 1995, 27, 1-15.
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Table 1. pKaValues for TPPS4, 8.0 µΜ in Acetate-Phosphate Buffer, 20 mM, Obtained by Spectrophotometric Titration in the Absence and Presence of Surfactants surfactant none CTAC SDS BRIJ-35 HPS
[surfactant] (mM) 20 40 20 20
pKa 4.52 2.56 4.70 2.80
quency, cycling through the decoupler list after each collection of 40 scans gained for each irradiation frequency. Multiplet peaks were irradiated, cycling the decoupler offset through the lines of the multiplet. Transient NOEs were measured using the same scheme as that for selective T1, the delay τ being in the range from 1 ms to 5 s. Typically, 40-720 scans were accumulated for each of the delays τ, cycling through the delay list after each collection of 40 scans gained for each delay. The postprocessing exponential filter with a line-broadening factor of 10 Hz (or 0.3-1 Hz in some pilot runs) was applied in the time domain. Results In the present study, the zwitterionic surfactants HPS and LPC, cationic CTAC, anionic SDS, and neutral Brij35 were used. Optical techniques provided precise stoichiometry and equilibrium constants for the reactions of protonation, self-aggregation, and binding to micelles in the low-concentration range of TPPS4. The sensitivity of NMR techniques to interatomic distances allowed us to obtain some spatial information on the TPPS4/surfactant system, albeit at higher concentrations. Optical Absorption Data. The absorption spectrum of TPPS4 in acetate-phosphate buffer, 20 mM, at pH 3.0 exhibits bands at 434 nm (Soret bands) and 645, 594, and 550 nm (Q bands) whereas at pH 7.5 the bands are centered at 414 nm (Soret bands) and 516, 552, 580, and 634 nm (Q bands). The considerable spectral changes as a function of pH are due to the protonation equilibrium of the pyrrole nitrogens in the center of the porphyrin ring. The absorption spectra were decomposed by the CCA algorithm as a sum of two components characteristic for the two species, protonated and deprotonated. The observed pKa in our buffer is 4.52, as shown in the Supporting Information. The addition of surfactants changed the absorption spectra of TPPS4 in both Soret- and Q-band regions, indicating its binding to surfactants. The pKa value of TPPS4 in acetate-phosphate buffer, 20 mM, changed in the presence of micelles (Table 1). Cationic micelles of CTAC were found to cause the most significant changes (almost 2-units decrease). Anionic micelles of SDS seem to cause a very slight, almost negligible, increase in pKa (Table 1), while nonionic micelles of Brij-35 induce a considerable decrease in pKa, almost as great as CTAC. The absence of pKa in HPS could be associated with the partial protonation of the sulfonate groups of the surfactant, which are in large excess as compared to porphyrin, in the used pH range. The addition of CTAC changes the position, width, and intensity of the bands in the absorption spectra of TPPS4 at both pHs (Table 2). The deconvolution of the spectra by the CCA method shows that, in the presence of cationic micelles of CTAC, TPPS4 at pH 3.0 does exist in solution as three species with relative concentrations depending on [CTAC] (Figure 1b,d). The first species characterized by λmax ) 434 and 645 nm (Figure 1b,d, species 1) has the
spectrum similar to that of the TPPS4 solution in the absence of CTAC. The content of this component decreases with the [CTAC] increase, and at a concentration ratio of [CTAC]/[TPPS4] ) 4:1 it practically disappears (Figure 1e,f, species 1). We can assign this component to the free TPPS4. The other two components exist only if CTAC is present in the solution. Thus, we can identify them as two different TPPS4 surfactant-bound species. The first one is characterized by an absorption spectrum with λmax ) 710 nm (Figure 1b,d, bound species I). Its Soret band has low intensity and is relatively broad (there seems to be some intensity at 490 nm which is relatively low) while a considerable red-shifted band is apparent at 710 nm. The content of this species at first increases with the [CTAC] increase and reaches a maximum at the same [CTAC]/[TPPS4] value where the free TPPS4 content becomes equal to zero; after this point it begins to decrease (Figure 1e,f, bound species I) in favor of a second bound species characterized by λmax ) 417 and 516 nm (Figure 1b,d, bound species II). At an excess of CTAC, the spectrum is represented solely by this component, showing that at a saturating concentration of surfactant this is the only species remaining in solution (Figure 1e,f, bound species II). For TPPS4 at pH 7.5, the deconvolution of the absorption spectra at different [CTAC] (not shown) as well shows the presence of three species, which should be assigned to one free and two bound TPPS4 species using the same arguments as those for pH 3.0. The content of the free TPPS4 species characterized by λmax ) 414 and 516 nm decreases with an increase in the [CTAC] and becomes negligible at [CTAC]/[TPPS4] ) 12:1. The content of the bound species I (λmax ) 517 nm) increases, reaches the maximum at [CTAC]/[TPPS4] ) 12:1, and then decreases with an increase in [CTAC] in favor of the TPPS4 bound species II (λmax ) 417 and 517 nm); at an excess of CTAC, only the bound species II of TPPS4 remains in the solution. The spectra of TPPS4 in the presence of zwitterionic micelles of HPS at high concentrations are similar to those at an excess of CTAC (Table 2). The analysis by the CCA algorithm demonstrates that, in the presence of HPS, only two TPPS4 species are present at either pH (3.0 and 7.5): the free species and the bound one, which is similar to the bound species II observed at CTAC excess. The bound species I was not observed in the presence of HPS. (For details, see the Supporting Information.) In the presence of anionic micelles of SDS, no significant spectral changes were observed in the whole range of surfactant concentrations and at both used pHs, save the little changes in the intensity of optical absorption bands. Fluorescence Experiments. The fluorescence spectrum of the TPPS4 in acetate-phosphate buffer, 20 mM, at pH 3.0 exhibited a band at 664 nm, while at pH 7.5 it was shifted to 641 nm. The addition of an excess concentration of surfactants changed the position of the maximum emission wavelength (Table 2) and the width of the band (not shown) in the fluorescence spectrum at both pHs. At pH 3.0 with cationic micelles of CTAC and zwitterionic micelles of HPS, the fluorescence maxima are shifted to shorter wavelengths, while anionic micelles did not produce any changes in the fluorescence maxima of the porphyrin. At pH 7.5, a small shift of the fluorescence maxima to the red wavelength region was observed with the exception of SDS, for which no changes occurred. Analysis of fluorescence data by the CCA method gave results similar to the ones obtained from absorption spectra. In the presence of CTAC at pH 7.5 (Figure 2a) the
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Table 2. Effect of the Micelles on the Wavelengths of Maximum Absorption, λmaxabs, and Maximum Emission, λmaxemi, of TPPS4 in Acetate-Phosphate Buffer, 20 mM, Obtained by Spectrophotometric and Spectrofluorimetric Titrationsa pH
buffer
CTAC
HPS
SDS
3.0
λmaxabs
434 645, 594, 550
417 516, 550, 587 643
417 516, 549, 588 643
434 645, 594, 557
7.5
λmaxemi λmaxabs
664 414 516, 552, 580 634 641
649 417 517, 549, 589 642 647
646 417 514, 548, 589 644 647
664 414 516, 553, 580 632 641
λmaxemi a
Surfactant concentrations are the same as those in Table 1; the TPPS4 concentration was between 7.8 and 9.6 µM.
Figure 1. Optical absorption spectra of TPPS4 in the presence of various CTAC concentrations in the acetate-phosphate buffer, 20 mM at pH 3.0. (a),(c) Soret and Q-bands, respectively, [TPPS4] ) 8.0 µM, cuvette path length l ) 0.4 cm, and 0 < [CTAC] < 5.0 mM. (b),(d) Deconvoluted spectra of TPPS4 species involved in the equilibrium; the maximum absorption wavelengths are shown for the components. (e) Relative contents of TPPS4 species as a function of [CTAC], species 1 (free porphyrin), bound species I (aggregated bound porphyrin), and bound species II (monomeric bound porphyrin); (f) Same as (e), expanded to show low surfactant concentration range.
coexistence of three species in solution was observed with relative concentrations depending on [CTAC]. Species 1 has an emission maximum centered at 641 nm and corresponds to the free porphyrin, the bound porphyrin at an excess of surfactant (bound species II) has the maximum emission at 647 nm, and the additional species at low surfactant concentration (bound species I) has a very low quantum yield of fluorescence (Figure 2b). At pH 3.0, the emission as a function of surfactant concentration behaves in a very similar way. The maximal fraction of this intermediate concentration species was observed at a surfactant/porphyrin ratio of 2:1 at pH 3.0 and 5:1 at pH 7.5. In the case of HPS, the deconvolution showed the presence of only two species in equilibrium: the free porphyrin and bound porphyrin. In the case of micelles of SDS no spectral changes were observed.
The addition of CTAC at low concentration reduced the TPPS4 quantum yield of fluorescence, as observed from the decrease in the integral fluorescence intensity (Figure 3); these changes are followed by a significant shift of the maximum of emission to the blue wavelength region at pH 3.0 and by a relatively smaller red shift at pH 7.5. Upon a further increase in [CTAC], the width of the spectrum decreased, the maximum shifted further to the blue wavelength region, and the integral fluorescence intensity increased again (Figure 3), this increase being greater at pH 7.5 as compared to pH 3.0. The minimum of the integral fluorescence intensity (J) represents the maximum content of the TPPS4 bound species I determined from the optical absorption and fluorescence emission spectra. Thus, our fluorescence data also demonstrate the presence of three species in TPPS4/CTAC
Interaction of TPPS4 with Ionic Surfactants
Figure 2. Fluorescence emission spectra of TPPS4 scaled in arbitrary units in the presence of various CTAC concentrations in the acetate-phosphate buffer, 20 mM at pH 7.5, λex ) 575 nm. (a) [TPPS4] ) 7.6 µM, cuvette path length l ) 1.0 cm, and 0 < [CTAC] < 1.7 mM. (b) Deconvoluted spectra of TPPS4 species involved in the equilibrium; the maximum emission wavelengths of components are shown. (c) Relative contents of TPPS4 species as a function of [CTAC], species 1 (free porphyrin), bound species I (aggregated bound porphyrin), and bound species II (monomeric bound porphyrin).
solutions: free porphyrin, bound porphyrin I, and bound porphyrin II, the bound I species having the minimum of fluorescence intensity. The recovery of fluorescence intensity at an excess of surfactant seems to be considerably higher at pH 7.5 (around 70%) as compared to pH 3.0 (around 40%). To monitor the effect of porphyrin concentration on the aggregation, a titration of TPPS4 with CTAC was performed at a concentration lower by an order of magnitude (Figure 3c,d). In this case, the integral fluorescence is still reduced because of aggregate formation, reaching a minimum at 12 and 16% of the initial fluorescence for pHs 3.0 and 7.5, followed by a recovery up to 74 and 189% at those pHs. The minimum of fluorescence is reached at a ratio surfactant/porphyrin of 25 at both pHs. Thus, our data suggest that, even at the lower porphyrin concentration, CTAC induces a decrease in fluorescence. In the case of HPS (data not shown), at pH 7.5 a 15% increase in total fluorescence was observed upon an increase in surfactant concentration, while at pH 3.0 an initial sharp decrease of total fluorescence to 44% of the initial value was followed by a recovery at higher surfactant concentrations up to 51% of the initial fluorescence. In this way, seemingly, for HPS there is a substantial quenching of total fluorescence at pH 3.0 that is not associated with porphyrin aggregation and could be due to the proximity of the sulfonate groups to the chromophore. At pH 7.5, the 15% increase in total fluorescence is indicative of binding of the porphyrin to the micelles and consequent slight reduction in porphyrin mobility.
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Light-Scattering Experiments. The scattered light intensity (SLI) of a solution in the absence of optical absorption depends on the wavelength as 1/λ4 (Rayleigh law). The buffer and surfactant solutions in the absence of porphyrin do not absorb in the spectral range studied; thus, scattered light (SL) spectra of solutions at different surfactant concentrations obeyed the Rayleigh law (Figure 4a, inset). The SLI slightly increases with the addition of surfactant. In the spectral range where a solution has optical absorption, an increased SLI can be observed as a result of the increase of the refractive index of the scattering medium in this range (resonance light-scattering (RLS) effect) (papers41,42 and references therein). Usually this increase is masked by absorption; however, when aggregates are formed, this effect can be strong enough since the RLS intensity is proportional to the square of the scattering particle volume. Thus, the RLS effect can be used as a test for aggregate formation.41,42 Figure 4b-d demonstrates the SL spectra of TPPS4 solutions in the presence of different CTAC concentrations. The SLI in the range between 400 and 500 nm initially increased dramatically as a result of the surfactant addition (the RLS effect) and decreased again upon further increase of [CTAC]. The data presented in Figure 4 correspond to pH 7.5; however, at pH 3.0 this effect is also observed. The SLI at 434 nm is shown in Figure 4e at pH 7.5 as a function of [CTAC]. The maximum increase was observed for a surfactant/porphyrin ratio of 17:1 at pH 7.5 and 4:1 at pH 3.0. These values are close to those obtained from optical absorption and fluorescence spectra analysis presented above for the maximum content of bound species I of the porphyrin, especially at pH 3.0. In the case of HPS, no RLS effect was observed upon titration of porphyrin solutions. Constants of Association to Micelles. In Table 3, the association constant (Kb) values obtained from the fits of the optical absorption of TPPS4 as a function of surfactant concentration to the binding equation are shown. The presented Kb values are the average values obtained through the absorbance measurements at several wavelengths for a number of experiments. For each surfactant, the association constants were determined both at pH 3.0 and 7.5 to obtain the association constants for the porphyrin at different levels of protonation. In the presence of cationic micelles of CTAC, at pH 3.0 we have a mixture of almost equal concentrations of protonated and deprotonated porphyrin, while at pH 7.5 only deprotonated porphyrin is present in the solution (see Table 1). The data at low CTAC concentration, significantly influenced by aggregation, were disregarded. (See the Supporting Information.) Since for CTAC the porphyrin aggregation is evident at both pHs, an additional binding experiment was performed with a 10-fold lower porphyrin concentration (8 × 10-7 M). In this case the binding constant at pH 7.5 remained the same, while at pH 3.0 the constant increased by a factor of 3. 1 H NMR Spectra. The incorporation of TPPS4 into the cationic micelles resulted in dramatic effects in the spectra of surfactants. The most noticeable effect in the spectra of CTAC was an upfield shift of the peaks belonging to the terminal methyl and neighboring methylenes of the hydrocarbon chain (see previous work20 and Supporting Information). There was some decrease observed in T1 for (41) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393-5399. (42) Pasternack, R. F.; Collings, P. J. Science 1995, 269, 935-939.
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Figure 3. Normalized integral of fluorescence spectra (J/J0) as a function of [CTAC]. J and J0 are integrals of fluorescence spectra in the presence and absence of CTAC, respectively. (a) pH 3.0, [TPPS4] ) 8.0 µM, (b) inset: expanded plot at low concentrations of CTAC; (c) pH 7.5, [TPPS4] ) 7.6 µM, (d) inset: expanded plot at low concentrations of CTAC; (e) pH 3.0, [TPPS4] ) 8.0 × 10-7 M; (f) pH 7.5, [TPPS4] ) 6.4 × 10-7 M.
all CTAC groups, seemingly because of the increase in their rotational correlation time τc. The effects induced by TPPS4 in the spectra of CTAC, HPS, and LPC were qualitatively similar (Figure 5). In the spectra of LPC, significant shifts were observed for the peaks of the hydrocarbon chain, and also appreciable shifts of the protons of the polar head, which is much larger than the head of CTAC. As opposed to the anionic TPPS4, cationic TMPyP incorporated only into anionic SDS micelles, producing similar effects on the terminal part of the SDS hydrocarbon chain, the interaction even with zwitterionic HPS and LPC being negligible. The spectrum of pure TPPS4 in solution (Figure 6a) is relatively invariant in the millimolar concentration range at pH above 6; at lower pH, the spectrum disappears.10 The appearance of two broad pyrrole peaks is usually attributed to a slow rate of tautomerism of the porphyrin central imine (-NH) protons.44 Also shown in Figure 6 are the changes occurring in the TPPS4 spectra upon its binding to micelles. The absence of changes in TPPS4 peak positions, when varying its concentration between 1 and 10 mM in the presence of 100 mM CTAC, indicated that the porphyrin is predominantly bound in these experimental conditions. The 1H NMR peaks of both CTAC and TPPS4 remained unchanged between pH 2.5 and 8. However, we observed the dication formation of TPPS4 in aqueous solution in the presence of the cationic CTAC (5 mM TPPS4/100 mM CTAC) at pH below 2.5, and in the presence of the zwit-
terionic LPC (10 mM TPPS4/20 mM LPC) at pH below 5. The transition to H2TPPS4 manifested itself in the disappearance of peaks belonging to the unbound fraction of TPPS4 as well as in smaller disturbances in surfactant chains. At the CTAC:TPPS4 molar ratio of 4:1, the solution turned to be essentially heterogeneous, with extremely broadened NMR lines. This phenomenon was observed even at pH as high as 5.6 and at a CTAC concentration of 40 mM (i.e., well above the cmc). Therefore, it was attributed to the formation of nonmicellar co-aggregates of CTAC and TPPS4. Proton T1 Relaxation Times. Both selective and nonselective T1’s of TPPS4 changed upon binding to micelles, the pattern of the alterations being different for CTAC and LPC (Table 4). The T1 values of TPPS4 bound to CTAC appeared to be pH-independent, i.e., the same for both states of protonation of TPPS4. In the conditions of this experiment, TPPS4 was predominantly bound to CTAC micelles, while in the presence of LPC the significant contribution of the free TPPS4 in solution should be taken into account. NOE Difference Spectroscopy. The location of TPPS4 monomers in CTAC and LPC micelles has been probed by (43) Rotenberg, M.; Margalit, R. Biochim. Biophys. Acta 1987, 905, 173-180. (44) Koehorst, R. B. M.; Hofstra, U.; Schaafsma, T. J. Magn. Reson. Chem. 1988, 26, 167-172.
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Figure 4. Spectra of scattered light in solutions of TPPS4, 1.7 µM at pH 7.5, and concentrations of CTAC in the range 0-20 mM. (a) Inset: spectra of solutions of pure surfactant in buffer normalized to that of pure buffer at λ ) 350 nm. (b)-(d) Spectra of solutions of porphyrin and CTAC showing the increase in scattering at low surfactant concentrations followed by its decrease at higher surfactant concentrations. (e) Intensity of resonance light scattering at λ ) 434 nm as a function of [CTAC]. Table 3. Association Constants Obtained by Fitting the Experimental Data from the Titrations of TPPS4 with Different Surfactants to the Binding Equilibrium Equation40 surfactant
pH
Kb (M-1)a
cmc (mM)a
CTAC
3.0 7.5
(10 ( 4) × 103 (32 ( 2) × 103
(50 ( 2) × 10-3 (66 ( 1) × 10-3
HPS
3.0 7.5
(26 ( 5) × 103 (15 ( 2) × 103
(50 ( 4) × 10-3 (18 ( 3) × 10-3
a K is the association constant; cmc is an adjustable parameter b from the binding equilibrium equation, as described in the Supporting Information.
NOE-difference spectroscopy. As can be seen from Figure 5, the signals of methylenes at different chain positions of surfactants become separated in the presence of TPPS4. Another important factor favoring signal quantitation is that the peaks for the surfactants and TPPS4 are situated in distinctly different parts of the spectral window. In slowly tumbling molecular aggregates, the steady-state NOE experiment is inappropriate, since spin diffusion spreads NOE uniformly throughout all participating protons. Therefore, the rate of NOE buildup was analyzed in truncated and transient experiments. Using the sample containing 100 mM CTAC in the presence of 5 mM TPPS4 at pH 5.0, the intensities of the TPPS4 peaks were measured upon irradiation of the CTAC resonances. In the truncated NOE mode (irradiation time below 400 ms), NOE was negligible when irradiating
Figure 5. 1H NMR spectra of 20 mM LPC ((a),(b)) and 20 mM HPS ((c),(d)) in D2O at pH 5.5 in the absence ((a),(c)) and presence ((b),(d)) of 10 mM TPPS4.
protons of the headgroup N-methyls and two methylenes adjacent to the nitrogen atom, while weak albeit measur-
6240 Langmuir, Vol. 15, No. 19, 1999
Figure 6. 1H NMR spectra of TPPS4 in aqueous solutions, 5.0 mM in TPPS4 at pH 7.7 (a) and 2.5 mM in TPPS4 and 100 mM in CTAC at pH 5.0 (b). Assignments: Ph, phenyl resonances; Pyr, pyrrole resonances.
able negative NOEs (about 3-5%) were found for other methylenes and terminal methyls of the hydrocarbon chain. Therefore, the transient NOE measurements (dynamics of changes in the TPPS4 peak intensity after selective inversion of the CTAC resonance) were carried out for all chain methylenes and terminal methyls. Figure 7 shows the NOE buildup and recovery curves when CTAC protons in different chain positions were inverted. The initial NOE buildup rates estimated from these curves are summarized in Table 5. It is clear that the buildup rates for both pyrrole and phenyl TPPS4 protons increase in the direction from the head to the end of the chain in the CTAC molecule when different CTAC methylenes are inverted. Yet the smallest buildup rate was found after inversion of the terminal methyl resonance. In different experimental design, the TPPS4 peaks were irradiated, while observing NOE at the CTAC peaks (the sample 10 mM in TPPS4 and 80 mM in CTAC, pH 5.6). It was impossible to increase the TPPS4/CTAC ratio, as desirable for this experiment, because of the strong aggregation. Although the measurements were thereby compromised by low effects and uncertainty in baseline correction, both in the truncated NOE mode (irradiation time 100-400 ms) and in the transient experiment, small negative NOE (absolute values of up to 2%) was observed for the chain protons of CTAC upon irradiation of any of the TPPS4 protons. For the TPPS4/LPC system, aqueous solutions, 8.3-10 mM in TPPS4 and 20 mM in LPC, were adjusted to pH 5.6, and the TPPS4 peaks were irradiated, while observing NOE at the LPC peaks. Here, in the truncated NOE mode, very small negative NOE values were observed for the chain methylenes adjacent to the carboxyl fragment and for the glycerol backbone of LPC, upon irradiation of any of the TPPS4 protons. In the transient NOE experiment, the highest NOE buildup rate was observed at the protons of the glycerol backbone and of the N-methyls. Yet reliable estimates of absolute values of the buildup rate for LPC failed because of the very short time scale comparable with the selective pulse duration. Discussion The results of this study, jointly with some earlier reports of others,30-32 show that charged porphyrins strongly interact with ionic micelles and, therefore, may not exclusively partition in aqueous compartments, as is generally assumed,29 but may also interact with cellular membranes. Very bulky porphyrin molecules significantly
Gandini et al.
disturb the molecular order in small micelles. The hydrodynamic radius of micelles is determined mainly by the size of the surfactant molecule that is well comparable with the size of the porphyrin, i.e., about 2 nm. The terminal parts of the surfactant hydrocarbon chains are mostly affected, according to the displacements of their NMR peaks (Figure 5, see the Supporting Information for CTAC). Similar effects have been observed also when other drugs with conjugated electron systems, such as papaverine, were incorporated into micelles.19 Upfield shifts in this spectral region are most probably due to the effect of the ring currents in the conjugated electron system of the porphyrin since the alternative possibility, the effect of charged groups, should lead to the higher polarity, which is usually associated with downfield shifts. The alterations in NMR spectra of TPPS4 upon its binding to micelles (Figure 6) also may indicate its location in a hydrophobic environment.30 Apparently, solubilization of porphyrins within nonpolar regions of micelles is determined, in general, by nonspecific hydrophobic interactions. However, when both molecules are charged, their interaction is significantly modulated by electrostatic factors. In particular, the anionic species (TPPS4 and its metal derivatives) were incorporated into cationic (CTAC and CTAB30), zwitterionic (LPC), and neutral (Brij-35 and Triton X-10030) rather than anionic (SDS) micelles. In contrast, the cationic species (TMPyP and its metal derivatives) were incorporated only into anionic SDS rather than cationic (CTAC and CTAB31), zwitterionic (LPC and HPS), and neutral (Triton X-10031) micelles. On the basis of the results presented in this study, we may conclude that, in the presence of CTAC, there exists an equilibrium between three species of TPPS4 in solution as a function of surfactant concentration: one free porphyrin species and at least two bound porphyrin species, one corresponding to monomeric bound porphyrin at a high surfactant concentration (bound species II) and another one corresponding to porphyrin-surfactant aggregates (bound species I). In conditions of optical experiments, TPPS4 aggregation was induced by CTAC at relatively low concentrations, a phenomenon already described for biomacromolecules (BSA and DNA).11-17 The exact TPPS4:CTAC stoichiometry depended on the pH and slightly varied according to the experimental technique, but at any rate, the aggregation occurred at a [CTAC] below 140 µM, i.e., below the cmc of about 1 mM. Upon an increase in the [CTAC], the aggregates decomposed in favor of the porphyrin-bound monomers, leading to a dramatic reduction in SLI. The issue of monomer-aggregate equilibrium of porphyrin derivatives in micellar systems has repeatedly been addressed. The propensity of TPPS4 to aggregate in acidic media both in homogeneous solution10,45,46 and in the presence of cationic surfactants32 because of its transition to dication (with fully protonated central nitrogens), (H+)2(H2TPPS4)4-, or briefly (H4TPPS4)2-, is well-known. According to our pH titration data, the pKa for dication formation of TPPS4 reduced from 4.5 in buffer to 2.5 in the presence of CTAC. The pKa shift to lower pH upon association with a cationic surfactant usually indicates complex formation between charged molecules.47,48 At pH 3.0, the maximum of the fraction of this aggregated species obtained from absorption spectra coincided with the minimum of integral fluorescence and the maximum of (45) Ribo´, J. M.; Crusats, J.; Farrera, J.-A.; Valero, M. L. J. Chem. Soc., Chem. Commun. 1994, 681-682. (46) Pasternack, R. F.; Schafer, K. F.; Hambright, P. Inorg. Chem. 1994, 33, 2062-2065.
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Table 4. Nonselective (T1ns) and Selective (T1sel) Relaxation Times (ms) of TPPS4 Protons in Homogeneous and Micellar Aqueous Solutions (Data Are Presented As Mean ( SEM; The Significance of the Difference between the Group Means Was Tested by the Two-Tailed Independent Student’s t test) pyrrole TPPS4, pH > 5 TPPS4+CTAC, pH 5a TPPS4+CTAC, pH 1a TPPS4+LPC, pH>6b TPPS4+LPC, pH 4.5b a
phenyl
T1ns
T1sel
T1ns
T1sel
330 ( 10 840 ( 30 970 ( 120 290 ( 20 460 ( 30e
280 ( 20 420 ( 20d 410 ( 20d 210 ( 40 210 ( 20d
790 ( 30 and 340 ( 870 ( 20 760 ( 40 530 ( 40 and 360 ( 40c 470 ( 10 30c
5 mM TPPS4/100 mM CTAC. b 10 mM TPPS4/20 mM LPC. c For o- and m-phenyl protons.
Figure 7. NOE buildup and recovery curves for the phenyl (a) and pyrrole (b) resonances of TPPS4 when CTAC methylene protons in different positions of the hydrocarbon chain were inverted. Black squares, methylene adjacent to the headgroup (3.39 ppm); white circles, methylenes in the beginning of the chain (1.41 ppm); white squares, in the middle of the chain (1.21 ppm); black circles, at the chain end (0.97 ppm). Table 5. NOE Buildup Rates (s-1) Observed on the TPPS4 Protons upon Inversion of Different 1H NMR Peaks of CTAC (Data Were Obtained in Three Independent Experimental Runs by the Transient NOE Technique Using D2O Solution, 5 mM in TPPS4 and 100 mM in CTAC, at pH 5.0) inverted peak of CTAC
phenyl of TPPS4
pyrrole of TPPS4
N-CH2 N-C-CH2 low-field peak of the (CH2)13 group in the middle of the (CH2)13 group high-field peak of the (CH2)13 group terminal CH3
-0.04 -0.05 -0.12 -0.21 -0.24 -0.02
-0.02 -0.09 -0.08 -0.19 -0.30 -0.01
RLS, being around 2-4 surfactants/porphyrin. In the presence of CTAC at this pH, the porphyrin exists as a mixture of (H4TPPS4)2- and free base (H2TPPS4)4- and is characterized by a significantly red-shifted Q band with maximum absorption at 710 nm, which has been associated to the formation of J aggregates (with horizontally displaced parallel molecular planes) of (H4TPPS4)2because of an edge-to-edge interaction. This is generally observed at porphyrin concentrations above 5 × 10-5 M32,45,46 and seems to be very pronounced at acidic pHs. At even higher porphyrin concentrations above 1 × 10-4 (47) Rabenstein, D. L.; Bratt, P.; Schierling, T.; Robert, J. M.; Guo, W. J. Am. Chem. Soc. 1992, 114, 3278-3285. (48) Yushmanov, V. E.; Perussi, J. R.; Imasato, H.; Ruggiero, A. C.; Tabak, M. Biophys. Chem. 1994, 52, 157-163.
d
770 ( 40 and 340 ( 40c 510 ( 10d 500 ( 40d 330 ( 10 and 170 ( 20c,d 240 ( 10d
p < 0.05 vs T1ns. e p < 0.05 vs pH > 6.
M, H aggregates (stacks) due to a face-to-face aggregation have been reported.32,45 The results of previous works on the interaction of TPPS4 with micelles in an aqueous medium30,32,33 are essentially in agreement with our present findings. It was shown that SDS does not interact with TPPS4, while both CTAB and Triton X-100 produce monomeric porphyrin through their interaction with the porphyrin in the high concentration range (above 3 × 10-4 M). However, the lack of pH control30 (which, as demonstrated in the present study, is an important factor) makes it difficult to justify some conclusions. An important role of the length of the alkyl chain of the surfactant in J aggregation of the TPPS4 in the presence of low concentrations of CTAB and other cationic surfactants was described at pH 3.5 and 8.0.32,33 The data at pH 3.5, however, do not refer to the pure dication (H4TPPS4)2-, as authors claimed,32 since the shift of the pKa of TPPS4 upon interaction has not been taken into account. Yet, their results are in agreement, in general, with our data, although the porphyrin:surfactant aggregation stoichiometry of 1:2 for CTAB is somewhat different. The relevance of the hydrophobic contribution to interaction has also been supported, since shorter chain surfactants are only able to induce aggregation at higher concentrations.32 A significant decrease in the fluorescence for the porphyrin/surfactant complex was reported,32,34,37 which is also similar to our findings. The formation of nonfluorescent species seems to be common for premicellar and self-aggregation of various ionic dyes in the presence of surfactants of opposite charge.22,34 Surfactants and other counterions may induce dimerization of free base ionic porphyrins.49 The free base (H2TPPS4)4- aggregates observed in the present study should be qualified as premicellar or nonmicellar porphyrin/surfactant complexes, also known as “dye-rich” mixed aggregates. The aggregation of porphyrins in aqueous surfactant solutions has been extensively studied by Whitten et al.34,35 Notably, the formation of slow equilibrating “dye-rich induced micelles” of picket-fence porphyrins and SDS (proven to be premicellar J aggregates) is not connected with the ring nitrogen protonation. For surfactants in the presence of porphyrins, it is reasonable to discriminate between cmc and critical aggregate concentration (cac). Although the cmc of surfactants is generally reported to be independent of the presence of porphyrins,21,33 the formation of premicellar mixed aggregates occurs at a cac below the cmc.33-35 The nonmicellar aggregation has much in common with the aggregation of TPPS4 on BSA12,14 and TMPyP on DNA.15-17 Probably, cationic micelles may reproduce to some extent the characteristics of the porphyrin binding sites on BSA (which are also positively charged). Indeed, in ref 12 TPPS4 is always in the free base form in (49) Chandrashekar, T. K.; van Willigen, H.; Ebersole, M. H. J. Phys. Chem. 1984, 88, 4326-4332.
6242 Langmuir, Vol. 15, No. 19, 1999
aggregates on BSA, without pH dependence in optical spectra between 4 and 8.5. The arrangement of TPPS4/CTAC aggregates may be different at pH 3.0 and 7.5. On one hand, although the light-scattering data at pH 7.5 show unequivocally the existence of aggregation, the red shift of the Q band was not observed, suggesting a different nature of the aggregates at this pH. In this case the porphyrin is entirely in the free base form (H2TPPS4)4-, implying a greater net porphyrin negative charge and a greater repulsion between porphyrin molecules. On the other hand, at pH 7.5 the number of surfactant molecules per porphyrin in the aggregates is about 3-4 times higher as compared to those at pH 3.0, suggesting that the aggregates should be greater. Also, the association constant estimated for TPPS4 with CTAC is a factor of 3 greater at pH 7.5 as compared to that at pH 3.0. The cmc formally obtained from the fitting of data (see the Supporting Information) is rather similar for both pHs (50-60 µM, Table 3), in agreement with the aggregation behavior well below the normal cmc for CTAC. Therefore, this value should be interpreted as the cac observed in the presence of TPPS4 below the cmc. Our data suggest that in the aggregates the delicate balance of porphyrin-porphyrin and porphyrin-surfactant interactions determines the nature of their molecular species in solution. In the recently appeared paper,50 using similar experimental approaches, the formation of two types of premicellar TPPS4/CTAB aggregates has been shown at pH 3.5, J aggregates at the porphyrin:surfactant ratio of 1:2, and H aggregates when porphyrin:surfactant ) 1:4. Only H aggregates were observed at pH 8.1. Taken together, those results50 and our present findings indicate that the J aggregates are ionic complexes of the (H4TPPS4)2- and two surfactant molecules as counterions. At higher surfactant concentration (porphyrin:surfactant ) 1:4) at pH 3.5, the elevated local concentration of cations induces the pKa shift of TPPS4 to lower pH, and the H aggregates are actually ionic complexes of the (H2TPPS4)4- with four surfactant molecules as counterions. The present findings with the zwitterionic surfactant HPS suggest that if the aggregation takes place at all, it is significantly less pronounced as compared to the cationic CTAC. Indeed, the optical absorption spectra, lightscattering data, and fluorescence emission do not indicate the formation of aggregates but just the existence of two states of the porphyrin: free in solution and bound to the micelles. Hence, the values of Kb should be consistent with those obtained by the inspection of the concentration of HPS where the fraction of bound porphyrin is 50% (this gives the value of the dissociation constant). At pH 7.5, both optical absorption (see the Supporting Information) and fluorescence (not shown) data give 1.4 × 104 M-1, a value very close to that obtained from the fit to the binding equation (Table 3). At pH 3.0, the value obtained from these curves is 1.2 × 104 M-1, which is lower by a factor of 2 as compared to the fit (Table 3). Since acidic pH would favor the free porphyrin aggregation, it is possible that at pH 3.0 some aggregation takes place, affecting the value of the apparent binding constant through preferential binding of monomers to HPS.13,43 The estimated association constants for HPS are quite similar to those for CTAC (Table 3), suggesting that the driving force for the porphyrin partition into the micelles is not associated directly with the electrostatic interaction; rather, it should involve immersion of the ring into the micelle interior. The pKa value observed for TPPS4 in the presence of nonionic Brij-35 (Table 1) also supports the relevance of the hydrophobic interaction in the surfactant-
Gandini et al.
porphyrin system. For HPS, the ratio of binding constants at pH 3.0 and 7.5 is opposite to that for CTAC (Table 3). The higher binding constant for HPS at pH 3.0 as compared to pH 7.5 may be due to the electrostatic attraction between the negative porphyrin and the HPS headgroup, which acquires some net positive charge due to the partial protonation of the sulfonate groups at pH 3.0. An electrostatic interaction of TPPS4 with ionizable headgroups of HPS is also in agreement with the TPPS4 fluorescence quenching observed in the presence of HPS. The NMR data confirm the general features of the ionization equilibria (e.g., the pKa shift) of TPPS4 in the presence of surfactants above their cmc and of its chargedependent interaction with micelles (e.g., lower disturbance of micellar structure with the decrease in electrostatic attraction). In addition, NMR provides information about relative localization in the TPPS4/surfactant complex. NMR techniques making use of spin-lattice relaxation phenomena (T1, NOE) are sensitive to interatomic distances and have been used to address the location of drugs in micelles and small lecithin vesicles.19,51,52 Therefore, we used those techniques to prove the location and aggregation of the porphyrin derivatives in micelles. Proton relaxation times T1 of a small ligand are often affected by intermolecular interactions more strongly than chemical shifts and are related to the molecular mobility of the corresponding chemical groups. Pathways of magnetic relaxation are different when only the signal of interest (selective relaxation) or all transitions in the spectrum (nonselective relaxation) were perturbed. The micelle protons are, seemingly, in the intermediate motional regime ω0τc ∼ 1 (ω0 is a Larmor frequency); in this case the selective and nonselective T1 do not differ significantly, as was shown earlier for CTAC19 and confirmed in this study for LPC (data not shown). However, for a small molecule undergoing significant immobilization, selective inversion of its spins may provide an additional index due to the different dependence of nonselective and selective spin-lattice relaxation rates on the correlation time τc.53-55 The ratios of selective and nonselective T1 values suggest that free TPPS4 is, seemingly, in an intermediate motional regime (ω0τc ∼ 1). In all experiments with micelles, the much shorter selective than nonselective T1’s of TPPS4 indicates slow molecular motion (ω0τc > 1) of TPPS4. It should be taken into account that in LPC, there is a fast exchange between the free and bound TPPS4 species, influencing apparent T1’s. However, solely immobilization and exchange with free species cannot explain differences between CTAC and LPC; there should be some specific intermolecular interaction resulting in different relaxation pathways for the two types of micelles. Contrary to FeTPPS4,20 no aggregates of TPPS4 on micelles of CTAC were detected: the formation of the dication at low pH resulted only in a lower degree of binding; however, both selective and nonselective T1 of the bound form are apparently pH (i.e., protonation-) (50) Maiti, N. C.; Mazumdar, S.; Periasamy, N. J. Phys. Chem. B 1998, 102, 1528-1538. (51) Kuroda, Y.; Fujiwara, Y. Biochim. Biophys. Acta 1987, 903, 395410. (52) Wakita, M.; Kuroda, Y.; Fujiwara, Y.; Nakagawa, T. Chem. Phys. Lipids 1992, 62, 45-54. (53) Valensin, G.; Kushnir, T.; Navon, G. J. Magn. Reson. 1982, 46, 23-29. (54) Bonechi, C.; Donati, A.; Picchi, M. P.; Rossi, C.; Tiezzi, E. Colloids Surf. 1996, A115, 89-95. (55) Yushmanov, V. E.; Tabak, M. J. Colloid Interface Sci. 1997, 191, 384-390.
Interaction of TPPS4 with Ionic Surfactants
insensitive in CTAC (Table 4). Unaltered T1 means unaltered molecular mobility and, hence, unchanged degree of aggregation. The aggregation of TPPS4 is, indeed, more difficult to follow by NMR as compared to FeTPPS4 since TPPS4 does not possess a paramagnetic ion. Yet it seems that our failure to observe TPPS4 aggregates on micelles is actually due to their different aggregation properties resulting from the different chemical nature of aggregates (oxygen bridge is possible only between metal ions), rather than due to any methodology flaw. Moreover, the formation of nonmicellar aggregates at the 4:1 molar ratio has also been observed well above the cmc. Obviously, at this ratio, there are no “normal” micelles anymore; there are nonmicellar aggregates of TPPS4 and CTAC, similar to those observed by optical techniques at low concentrations. NOE between TPPS4 and surfactant molecules indicates incorporation of the porphyrin into micelles and molecular contact between them. Interpretation of the NOE data requires certain caution since the NOE buildup rate depends on both interatomic distance and correlation time τc for molecular motion, which is different in different parts of the micelle. Particularly, lower NOE for the terminal CH3 of the hydrocarbon chain may be due to its higher mobility. Mobility of other hydrogens in the chain is roughly equal, decreasing slightly in the direction from tail to head. Therefore, our chemical shift and NOE data for TPPS4/surfactant complexes (Figures 5 and 7, Table 5, and the Supporting Information) indicate that TPPS4 is located mainly in the hydrophobic core in CTAC micelles, while in LPC its involvement in the polar area is much larger. Again, one should be cautious about increased NOE buildup rates on the glycerol backbone protons of LPC: they might be due in part to their low mobility. However, N-methyl protons of LPC also had large NOE in a transient experiment. The difference in location may also account for the different T1 behavior of TPPS4 in CTAC and LPC micelles (Table 4).
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Though the incorporation of TPPS4 in micelles has mainly a hydrophobic nature, different location and affinity of TPPS4 in various micelles may be rationalized through electrostatic factors. Indeed, the binding constants for CTAC and HPS have the same order of magnitude (Table 3). However, the degree of interaction of TPPS4 with hydrocarbon chains of LPC is significantly lower as compared to those of HPS and CTAC (as has been proven by comparing NMR spectra at the same TPPS4/surfactant ratio, Figure 5). When the nearest charge is positive (in CTAC and HPS), it may stabilize the bulky porphyrin molecule inside the hydrophobic layer. By contrast, in LPC the nearest charge is negative; it may have a destabilizing effect on the porphyrin, repulsing and displacing the latter in the direction of the nearest positive charge, i.e., to the outer part of the polar head. Further experiments with micelles are underway to clarify the nature of the porphyrin interaction with biological structures, complementing the data obtained on proteins and DNA. Chemical modification of metalloporphyrins altering their aggregation properties, may serve as a fine tool for influencing their therapeutic efficiency and biodistribution. Acknowledgment. This work was supported by CNPq, FAPESP, and FINEP. V.E.Y. and I.E.B. were supported by visiting grants from CNPq. Supporting Information Available: Chemical structures and micellar parameters of the porphyrin and surfactants; routines of sample preparation and determination of pKa and binding constants; UV-vis spectra of TPPS4 as a function of pH and concentration of HPS and their analysis by CCA; typical fits of UV-vis data to the binding equation; 1H NMR spectra of CTAC in the presence of TPPS4. This material is available free of charge via the Internet at http://pubs.acs.org. LA990108W
