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Steroidal surfactants: detection of premicellar aggregation, secondary aggregation changes in micelles, and hosting of a highly charged negative substance Ramon Barnadas-Rodríguez, and Josep Cladera Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01352 • Publication Date (Web): 05 Aug 2015 Downloaded from http://pubs.acs.org on August 11, 2015

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Steroidal surfactants: detection of premicellar aggregation, secondary aggregation changes in micelles, and hosting of a highly charged negative substance Ramon Barnadas-Rodríguez*, Josep Cladera Centre d’Estudis en Biofísica, Unitat de Biofísica, Departament de Bioqímica i Biologia Molecular. Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Cerdanyola, Catalonia, Spain. Corresponding author Ramon Barnadas-Rodríguez Centre d’Estudis en Biofísica, Unitat de Biofísica, Departament de Bioqímica i Biologia Molecular. Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Cerdanyola, Catalonia, Spain. E-mail: [email protected] Tel: 0034935868476 Fax: 0034935811907

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ABSTRACT CHAPSO and CHAPS are zwitterionic surfactants derived from bile salts which are usually employed in protein purification and for the preparation of liposomes and bicelles. Despite their spread use, there are significant discrepancies on the critical concentrations that determine their aggregation behavior. In this work, we study the interaction between these surfactants with the negative fluorescent dye pyranine (HPTS) by absorbance, fluorescence and infrared spectrometry to establish their concentrationdependent aggregation. For the studied surfactants, we detect three critical concentrations showing their concentration-dependent presence as a monomeric from, pre-micellar aggregates, micelles and a second type of micelles in aqueous medium. The nature of the interaction of HPTS with the surfactants was studied using analogues of their tails and the negative bile salt taurocholate (TC) as reference for the sterol ring. The results indicate that the chemical groups involved are the hydroxyl groups of the polar face of the sterol ring and the sulfonate groups of the dye. This interaction causes not only the incorporation of the negative dye in CHAPSO and CHAPS micelles, but also its association with their pre-micellar aggregates. Surprisingly, this hosting behavior for a negative charged molecule was also detected for the negative bile salt TC, bypassing, in this way, the electrostatic repulsion between the guest and the host.

Keywords: CHAPSO, CHAPS, HPTS, pyranine, tensioactive.


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INTRODUCTION CHAPSO and CHAPS are zwiterionic surfactants usually employed for protein purification1-3 and crystallization,4,5 and liposome and bicelle preparation or solubilization.6-8 Their singular properties as surfactants are a consequence of their particular structure. They are derived from bile salts and also contain a tail with a dipolar sulfobetaine head group with one positive and one negative charge. Their steroid ring structure confers to both surfactants the characteristics observed in planar molecules with a hydrophilic and a hydrophobic face. Furthermore, their tail gives to CHAPSO and CHAPS a large isoelectric pH range and a mild detergent character. The presence of three hydroxyl groups in the sterol ring allows establishing a parallelism with the behavior of some bile salts such as cholic acid, glicocholic acid and, particularly, taurocholate (TC), which also contains a sulfonate group in its tail. The aggregation mechanism of bile salts has been extensively studied. They show a non classical surfactant aggregation where micelles are formed from a well determined concentration. On the contrary, they show a broad range of critical micellar concentration (CMC) which, depending on the proposed mechanism, involves a slow size growth of the surfactant aggregates or reveals the existence of some critical points during the slow aggregation process.9-13 In both cases, there is an initial formation of small aggregates driven by the hydrophobic interaction between the apolar faces of the rings of two molecules (back-to-back aggregation). Literature shows a great variety of the CMCs values6, 10, 11, 14-18 that can be explained either by the slow change of the successive aggregation steps or by the experimental constraints. Regarding CHAPSO and CHAPS, literature about their aggregation properties is scarce, and it is basically centered on CHAPS. As in the case of the three hydroxylated bile salts previously mentioned, the back-to-back association and a slow growth of the CHAPS aggregates


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are proposed,9, 19, 20 although changes in the micellar stage are also reported.21 Again, as in the bile salts, a broad range of CMCs (from 4.6 to 32 mM) is detected6, 9, 19-22 where NMR, chromatographic and fluorescence techniques are employed. Fluorescence measurements are useful because of their high sensitivity and also because at the same time allow for evaluation of the interaction of model molecules with the surfactant aggregates.13, 23-25 The characterization of this interaction is important since from its knowledge it is possible to know how micelles act as hosting systems, that is, we are able to characterize their capability to entrap other substances and, complementary, to determine what kind of molecules can become their guests. Among the fluorescent molecules used, pyranine (HPTS) has shown to be a good dye to not only detect the CMC of some positive and neutral surfactants, but also the formation of pre-micellar aggregates.26 The use of this dye is based on its excited species, which respond differently to the different types of aggregates formed by the surfactants. The initial aim of the present work was to study the mechanism of aggregation of CHAPSO and CHAPS to elucidate if pre-micellar aggregates are formed. This objective was accomplished but additional relevant information is presented in the paper. Several substances were used as model molecules to determine how the interaction between the dye and the surfactants was produced. One of them was TC, and the results also allowed us to characterize the behavior of this negative bile salt. Finally, some unexpected conclusions are drawn about the hosting behavior of CHAPSO, CHAPS and TC.

METHODS Materials Pyranine (HPTS), CHAPSO, CHAPS, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), and sodium taurocholate (TC) were from Sigma-Aldich. 2-hydroxy-3trimethylammonium-1-propanesulfonate (HAPS) was a gift of Dr. Joan Suades.27 The 4 ACS Paragon Plus Environment

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molecular formula of this product is HAPS·3.4NaCl.The compounds used are shown in Figure 1. Sample Preparation All solutions were obtained using purified water (resistivity ≥ 15 MΩ·cm). The absorbance spectrum of HPTS exhibits important changes at a pH higher than 6,

Figure 1. Chemicals used in the study.

and the fluorescence emission spectrum of HPTS shows no variation between pH 3 and 7.28 To avoid the effect of small pH changes on the absorbance and fluorescence spectrum of HPTS, the pH of all the samples was adjusted at 5 using NaOH or HCl stock solutions. Absorbance measurements Absorption spectra were obtained from 350 to 450 nm at a resolution of 0.5 nm using a Varian CARY 3Bio spectrophotometer. Sample temperature was kept at 25ºC under magnetic stirring. The spectra were smoothed (Cary WinUV 2.00 software) to improve the determination of the position and absorbance of the main peak of HPTS. Fluorescence measurements and analyses Fluorescence spectra were acquired with a PTI QuantaMaster spectrofluorimeter, termostated at 25ºC and with magnetic stirring. Samples were excited at 350 nm, and the emission spectra were recorded from 360 to 650 nm with a resolution of 1 nm. Usually, HPTS concentration was 6 µM, but also 0.6 and 60 µM HPTS were used to determinate whether or not the dye influenced the aggregation of the tensioactives. To minimize the inner filter effect of the 60 µM HPTS sample we used a 1-mL rectangular 5 ACS Paragon Plus Environment

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cuvette (10 mm x 3 mm) and the shorter pathlength was perpendicular to the excitation beam. The spectra were smoothed (PTI FeliX32 analysis software), their baseline adjusted to zero and the component bands of the peaks were calculated by deconvolution (Grams 3.2, Galactic, Salem, NH). The minimum quantity of overlapping Gaussian curves was obtained with a nonlinear least-squares multipeaks fitting procedure. Thus, the existence of component bands which would only contribute to a non-necessary continuous decrease of the statistical residuals was avoided. Fourier transform infrared attenuated total reflection spectroscopy (ATR-FTIR) 5.6 to 22.4 uL of stock sample aqueous solutions were carefully dropped on the central part of a Ge crystal and placed for 30 minutes into a desiccator connected to a vacuum pump. Afterwards, the films were flushed with dry nitrogen for 1 minute. The spectra (from 700 to 4500 cm-1) were acquired with a Fourier transformed infrared spectrometer (Varian 7000e FT-IR) at a resolution of 2 cm-1. 300 to 1000 scans were accumulated.

RESULTS Effect of CHAPSO on the spectral properties of the ground state of HPTS At pH 5 and in absence of CHAPSO, the main species of HPTS (pKa = 7.4) in the ground state is the protonated form (ROH), the dye bears three negative charges and its absorbance peak is

Figure 2. Position of the maximum of absorption of HPTS 6 μM (ROH species) at pH 5 as a function of the CHAPSO concentration (mean ± std; n=3).

located at 403.2 ± 0.5 nm (n=3). The presence of CHAPSO modified the absorption spectrum of 6 µM HPTS (see SI1 in the Supporting Information) and a significant bathochromic displacement of its maximum was observed (Figure 2). In addition, the absorbance values showed minor variations. The onset of the shift in the absorbance


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spectrum was located at 2-4 mM of CHAPSO. The shift reached a plateau at a surfactant concentration of 20-40 mM, and the peak became centered at 406.7 ± 0.4 nm. No detection of the deprotonated form (RO-) of HPTS was observed.

Figure 3. a) Percentage ratio of the fluorescence emission of HPTS 0.6 (red circle, n=2), 6 (green square, n=4; and 60 μM (blue triangle, n=2) at 509 and 439 nm at pH 5 as a function of CHAPSO concentration (mean ± std). Inset: fluorescence emission spectra of HPTS at pH 5 at 0 (line), 13 (dashed) and 40 mM (dotted) of CHAPSO. The two * -* peaks, centered at 439 and 509 nm, correspond to the ROH and the RO species respectively. b) Position of the -* two fluorescent emission maxima of ROH* (black circle) and RO (light red square) species at pH 5 as a function of the CHAPSO concentration (mean ± std, n =4).

Effect of CHAPSO on the spectral properties of the excited states of HPTS In absence of the surfactant, a solution of 6 µM HPTS exhibited its well-known emission spectrum which contains two peaks (inset Figure 3a). One peak is centered at 509 nm and corresponds to the RO-* form, that is, excited HPTS (HPTS*) with 4 negative charges. The second peak is caused by the emission of the ROH* species (HPTS* with 3 negative charges) and is located at 439 nm. In the absence of CHAPSO and at pH 5 the main form of HPTS* is the deprotonated species since the prototropic equilibrium of the photoacid has a very low pK*a, of about 1.4.28, 29 Upon increasing the concentration of CHAPSO, the emission spectrum of HPTS showed two types of modifications. First, there was a gradual decrease of the intensity of the RO-* peak and an increase of the ROH* peak. Therefore the formation of ROH* was favored. This phenomenon was monitored by the ratio of the fluorescence intensities at 509 and 439 nm, and expressed as the percentage of the ratio obtained in absence of CHAPSO (Figure 3a). The ratio was also calculated at 0.6 and 60 µM HPTS, and there were no 7 ACS Paragon Plus Environment

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significant differences (Figure 3a). The second type of variation in the fluorescence spectrum of HPTS observed in presence of CHAPSO was a shift of the maxima of the peaks (Figure 3b). A concentration

Figure 4. Experimental spectra and component bands of HPTS 6 μM in water at pH 5 at (a) 0 and (b) 40 mM of CHAPSO.

of CHAPSO higher than 0.3 mM caused a hypsochromic shift of the maximum of the ROH* species. It shifted almost 12 nm from 439.1 ± 0.8 nm (n=4), in absence of CHAPSO, to 427.4 ± 0.5 nm (n=4) at 40 mM of CHAPSO. The behavior of the RO-* form was opposite. A small bathochromic shift from 508.5 ± 0.5 nm to 512.2 ± 0.5 nm (n=4) was detected for CHAPSO concentrations progressively higher than 8 mM. Thus, despite the relative high experimental error, it can be observed that the peaks of both species did not shift simultaneously being also affected in a different way. As can be observed in the inset of Figure 3a, the peaks of ROH* and RO-* are partially overlapped. Thus, the quantification of the relative amount of both species, by means of their corresponding maximum intensities, is significantly altered by this fact. To circumvent this problem, the component bands of the emission spectra of HPTS* were obtained. Figure 4 shows two examples that correspond to the lower and higher concentrations of CHAPSO used in the present work. The number of component bands (see SI2 in the Supporting Information) and their maxima can be associated to different sub-ranges of concentration of the surfactant (see SI3 in the Supporting Information). The area of the ROH* and RO-* peaks were obtained from the areas of the component bands, whereas their values were expressed as the percentage of the area obtained in absence of the surfactant, that is, the percentage of quantum yield was calculated (Figure 5). As can be observed, the relative quantum yield of HPTS* was constant from 8 ACS Paragon Plus Environment

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0 to, approximately, 10 mM of CHAPSO (inset Figure 5). Since in this range we also observed changes in the fluorescence of the ROH* and RO-* species, the constant value of the quantum yield indicates that the presence of the surfactant only modifies the equilibrium between both forms. At CHAPSO concentrations higher than 14 ± 3 mM (obtained from the two closest points that include the break point), a minor but

*

*

Figure 5. Relative quantum yield of HPTS (red circles), ROH -* (green squares) and RO (blue triangles) species in water at pH 5 as a function of CHAPSO concentration (mean ± std, n ≥ 3). Inset: detail of the % Quantum Yield range from 90 to 107 %. Quantum yield was obtained as the percentage ratio of the areas of the corresponding component bands over the total area of the dye in absence of the surfactant.

significant increment of the total fluorescence of HPTS* was detected. The fluorescence of the RO-* form had a very slow decrease from 0 to 4-6 mM of CHAPSO, and a subsequent increase of its concentration caused a sharp drop of the fluorescence. Since the CMC of CHAPSO is considered to be between 6 and 8 mM, both phenomena, the strong decrease in the RO-* fluorescence and the small HPTS* fluorescence increment, can be attributed to the interaction of HPTS with different CHAPSO aggregates. On the other hand, a significant and gradual increment in ROH* quantum yield was detected for CHAPSO concentrations higher than 0.3-1 mM. This fact surprisingly reveals that the formation of the ROH* form is favored at a bulk pH which is more than three times greater than the pK*a. Thus, it is clear that the changes in the relative quantum yield of ROH* and RO-* take place at different CHAPSO concentrations, revelating the existence of different kind of environments caused by the surfactant. One of the most useful and common ways to characterize fluorescence changes of fluorophores caused by other substances, as quenchers, is the use of the Stern-Volmer 9 ACS Paragon Plus Environment

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relationship:

1 . 1

where F0 is the intensity fluorescence in absence of the quencher, F is the intensity in presence of the quencher at a given concentration [Q], and K is the Stern-Volmer constant. It is obvious that, if needed, the intensity can be substituted by the area (A) of the corresponding peak. To characterize the effect of CHAPSO on HPTS fluorescence, the area ratios of ROH*,

Figure 6. Stern-Volmer plot of HPTS fluorescence as a function of CHAPSO concentration at pH 5 calculated from the from * * area ratio (mean ± std, n = 5) of HPTS (red circles), ROH -* (green squares) and RO (blue triangles). Inset: plot of the -* * ratio of fluorescence areas (A) of RO and ROH species at three CHAPSO concentrations (red circle: 0 mM; green squares: 4 mM; blue triangles: 40 mM) as a function of pH (mean ± std, n = 3).

RO-* and HPTS* were obtained and plotted as a function of the surfactant concentration (Figure 6). From these results, four main observations can be pointed out. First, the RO*

form shows an apparent quenching (remember that no real quenching of HPTS* is

detected in Figure 5), but that it does not fit Equation 1. Effectively, a critical concentration of CHAPSO is needed to start decreasing the RO-* fluorescence (increase of A0/A), which is clearly identified as a break point. Consequently, the curve of the apparent quenching reaches a value of 1 at a (critical) CHAPSO concentration higher than 0. This critical value can be calculated from the intersection between the regression curve obtained with the points corresponding to the fluorescence decrease with value A0/A = 1. The concentration obtained was 6.57 ± 0.52 mM (n= 5), which is compatible with the classical CMC of CHAPSO. Therefore, it can be stated that the formation of micelles strongly reduces the formation of the RO-* form when the ground state of the HPTS becomes excited by its interaction with the surfactant. Or, from another point of 10 ACS Paragon Plus Environment

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view the excited form of HPTS with four negative charges is a good probe to monitor the formation of micelles (CMC1) of a zwitterionic surfactant. Second, the ROH* form exhibits an increase in fluorescence (that is, it has a negative K value) and, as in the case of RO-*, a critical concentration of CHAPSO is detected. Using the same procedure as that used for the RO-* species, we obtained a value of 0.69 ± 0.10 mM (n=5). This critical value, named pre-CMC1, and summarized with the rest of parameters derived from the Stern-Volmer plots in SI4 (Supporting Information), is smaller than the CMC1 and, consequently, indicates an aggregation state of the surfactant which appears before the formation of micelles, that is, it is an evidence of the existence of pre-micellar aggregates. Third, the ROH* curve shows an important subsequent change at high CHAPSO concentrations. The value of the corresponding break point was 11.4 ± 0.7 mM (n=5) (shown as CMC2 in SI4). And fourth, the Stern-Volmer plot for HPTS*, that is, the ratio obtained from the total area of the emission spectra, shows a break point at 14.5 ± 1.6 mM (n=5) (SI5), a value which is similar to the CMC2. As stated above, at pH 5 the different microenvironments originated by CHAPSO noticeably promote the ROH* species in a bulk that favors the existence of the RO-* form. To better characterize the nature of the interaction between the tensioactive and the dye, a fluorescent titration of HPTS was performed at several CHAPSO concentrations. The ratio of the areas of each excited species obtained from the spectra was plotted versus the bulk pH. As can be seen (inset Figure 6), in absence of the surfactant and even at low pH, RO-* was the main species, and its relative amount rapidly increased with the pH, reaching a plateau at, approximately pH 4. In these conditions, 98% of the fluorescent signal (50:1 area ratio) was caused by the RO-* form. The ratio could not be efficiently calculated at a pH higher than 8 because of the extremely low fluorescence of ROH*. Taking into account the pre-micellar 11 ACS Paragon Plus Environment

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concentration previously obtained (0.69 ± 0.10 mM), at a CHAPSO concentration of 4 mM more than 80% of the molecules would constitute pre-micellar aggregates and, consequently, the fluorescence spectra of HPTS obtained by the titration would be mainly a consequence of the interaction of the dye with those *

structures. At this surfactant concentration, and between pH 4 and 7, the relative

*

Figure 7. Relative quantum yield of HPTS (circles), ROH (squares) and RO-* (triangles) in water at pH 5 as a function of CHAPS (black) and TC (light red) concentration (mean ± std, n =2). Inset: detail of the % Quantum Yield range from 94 to 110%.

quantity of the ROH* form was higher than that obtained in the absence of CHAPSO. Now, 95% of the total area was originated by the RO-* species. Finally, between pH 2 and 8, and at 40 mM of CHAPSO (when more than 80% of these molecules constitute micelles) the relative area of RO-* decreased to 60%. Thus, in this pH range, the presence of micelles of CHAPSO noticeably increased the relative amount of ROH* from 2 (0 mM CHAPSO) to 40 % (40 mM CHAPSO). Effect of CHAPS and TC on spectral properties of HPTS We now investigate the effect of CHAPS and TC on HPTS fluorescence. CHAPS has a similar structure to CHAPSO but it has no hydroxyl group in the tail (Figure 1), whereas TC is a negative bile salt with a similar structure to CHAPSO and CHAPS. As shown in SI5 (Supporting Information), CHAPS showed the same effect on the ground state of the fluorescent dye as CHAPSO. In the same figure it can be observed that TC slightly modified the absorption spectrum of HPTS at high concentrations. Concerning the modification of the fluorescent spectrum of the fluorophore, both molecules caused a decrease of the fluorescence ratio between the excited species (see SI6 in the


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Supporting Information) and also caused a shift of the ROH* peak (see SI7 in the Supporting Information). However only CHAPS shifted the maximum of the RO-* peak (see SI8 in the Supporting Information). As in the case of CHAPSO, in the presence of CHAPS and TC there was a displacement of the equilibrium of HPTS*, no true quenching was observed (Figure 7),

*

-*

Figure 8. Stern-Volmer plot of ROH (squares) and RO (triangles) fluorescence as a function of CHAPS (black) and TC (light red) concentrations at pH 5 (mean ± std, n = 2). Inset: detail of the area ratio range from 0.98 to 1.03.

and the total fluorescence of HPTS* showed a slow increment (inset Figure 7). The relative quantum yield and fluorescence of the RO-* form decreased in TC solutions for a concentration of the bile salt higher than 10 mM (inset Figure 7). From the curves in Figure 8 the next critical concentrations were calculated (SI4): CHAPS (n=2), 0.59 ± 0.06 mM, 5.07 ± 0.64 mM and 11.4 ± 0.2 mM; TC (n=2), 2.72 ± 0.32 mM, 8 ± 2.3 mM. A break point was also observed in the HPTS* area ratio of CHAPS and TC, and critical concentrations of 8 ± 2 mM and 15 ± 5.8 mM were obtained respectively. Effect of CAPS and HAPS on the spectral properties of HPTS Due to the fact that the structure of HAPS and CAPS are similar to the tails of CHAPSO and TC, respectively, their effect on the spectral properties of HPTS was investigated. The only consequence observed was the change of the ratio of the fluorescence at 509 and 439 (SI6). An increment was produced in both cases, that is, an opposite effect to that caused by CHAPSO, CHAPS and TC. This phenomenon can be explained by the presence of NaCl, since it is known that Na+ favors the formation of the RO-* species.28 ATR-FTIR spectroscopy


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Figure 9 shows the spectra of four different samples. One of them is the spectrum of pure CHAPSO, which is very similar to that of CHAPS and TC (not shown) because of their analogous structure. The most relevant absorption bands and

Figure 9. Infrared FT-ATR spectra of CHAPSO (red continuous line), HPTS (black long dashed line), experimental mixture of CHAPSO and HPTS at 1:0.5 molar ratio (blue dotted line) and synthetic mixture of CHAPSO and HPTS at 1:0.5 molar ratio (green short dashed line).

peaks for these three surfactants were: 3700-3000 cm-1, OH and NH stretching (st); 3000-2780 cm-1, CH2 and CH3 st; 1650 cm-1, amide I CO st; 1550 cm-1, amide II NH bend (δ) and CN st; 1500-1430 cm-1, CH2 and CH3 δ (1480-1430 cm-1 for TC); 1373 cm-1, OH δ in plane; 1250-1150 cm-1, SO st; 1077 cm-1, CH-OH st . The second spectrum was obtained with pure HPTS and exhibits an important overlap with that of CHAPSO. Four main ranges can be considered in it: 3700-3000 cm-1, OH st; 1250-1100 cm-1, SO st; 1100-1000 cm-1, SO3- st; and 1650-1250 cm-1, which includes C-O st and aromatic ring deformations.30 A small peak of water at 1650 cm-1 (presumably strongly bounded to the sulfonate groups) was also obtained for all the performed replicas. The third spectrum shown was acquired from a film made with a mixture of CHAPSO and HPTS at a 1:0.5 molar ratio. The film contained the same amount of both molecules that was used for obtaining the corresponding pure spectra. The form and position of the peaks of the experimental spectrum of the mixture should be indicative of the existence of interaction between HPTS and CHAPSO. The last spectrum, the so-called synthetic, was obtained mathematically by the addition of the pure spectra of HPTS and CHAPSO. However, before adding the pure spectra it is necessary to use a correction a factor for each pure spectrum. For the case of CHAPSO (CHAPS and TC), the factor


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was obtained using the peaks that ranged from 3000 to 2780 cm-1 after a baseline correction. These peaks did not shift in the experimental spectrum of the mixture and, consequently, their area has to be the same than that

Figure 10. Difference spectra between the synthetic and the experimental spectra of CHAPSO (black continuous line), CHAPS (red long dashed line) and TC (green short dashed line) mixtures with HPTS at 1:0.5 molar ratio.

in the pure spectrum. Thus, the ratio of the corresponding areas allows us to calculate the correction factor for the mathematical addition. The same method was applied for obtaining the factor for the HPTS spectrum using the peaks located at 790 and 712 cm-1. Figure 9 contains the pure spectra after employing the correction factor and, therefore, they can be compared with the experimental and synthetic ones. The peaks with major changes are indicative of the interaction between HPTS and CHAPSO. For a better analysis the difference spectrum of the previous spectra was obtained and calculated for 1:0.5, 1:0.25 and 1:0.125 CHAPSO/HPTS molar ratios. The results (not shown) indicate that there is a concentration dependent effect of HPTS on the changes produced in the spectra and that these modifications affect in a very intense manner the OH and SO3- regions (Figure 10). Surprisingly, there was no significant alteration of the amide I and amide II bands of CHAPSO and, in this region, only the peaks of HPTS showed a modification. Finally, the same type of difference spectra was obtained for mixtures of CHAPS or TC with HPTS at a 1:0.5 molar ratio, and the results (Figure 10) show that the same chemical groups as that found for the case of CHAPSO/HPTS mixtures were involved.

DISCUSSION Interaction of ground state HPTS with CHAPSO and CHAPS 15 ACS Paragon Plus Environment

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The effects of CHAPSO and CHAPS on the spectral properties of HPTS were almost the same. The modification of the absorbance spectrum of the dye (Figure 2, SI5) evidences that, in the ground state, HPTS experiences a change of environment driven by the formation of aggregates of both surfactants. However, this modification is not strong enough to cause a displacement of the equilibrium between the ROH and ROspecies. The reported effect can be better quantified when it is compared with that caused by other surfactants. In this way, the non-ionic Triton X-100 interacts weakly with HPTS, and this interaction can be monitored by fluorescence methods but it is not detected by absorbance spectroscopy.26 It is also known that the strong interaction between HPTS and different kinds of aggregates of the n-alkyl trimethylammonium bromides family (positive charged), intensely modifies the absorbance spectrum of the dye.26 This interaction causes not only a shift of the maximum of the ROH species but produces also absorbance variations. Thus, although CHAPSO and CHAPS do not bear any net charge, the observed shift of HPTS and the small absorbance variations indicate that there is a significant interaction of the dye with CHAPSO and CHAPS aggregates. No further information on the nature of the interaction could be obtained but, on the contrary, the fluorescent changes caused on HPTS provided more detailed information. Aggregation states of CHAPSO and CHAPS detected by HPTS fluorescence The fluorescence results shown in Figure 3a indicate that HPTS did not influence the aggregation state of the surfactants, at least, within two orders of magnitude of the dye concentration (from 0.6 to 60 µM). Thus, the observed changes can be interpreted as a consequence of the aggregation state/s of the surfactants on the dye, being these aggregation states are not induced by the presence of HPTS. It is in this sense that HPTS can be considered as an impurity with no effect on the micellization process such as, for example, pyrene in the work of Matsuoka and Moroi.11 In fact, in the present 16 ACS Paragon Plus Environment

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work, the minimum critical concentration of surfactant detected (about 0.6 mM for CHAPS) is 1000 times higher than the minimum concentration of HPTS used in the experiment, and even the same results were obtained when the experiments were performed at a dye concentration 100 times higher. However, we should contemplate the possibility of a simultaneous formation of HPTS-aggregate structures and other structures not being probed. Despite this scenario could be possible, it can be considered unlikely if we take into account that, in principle, the dye should interact randomly with any of the aggregate structures that the surfactants form. Moreover, this assumption is reinforced by the fact that the classical CMCs obtained in our work for CHAPSO, CHAPS and TC are compatible with those indicated in the literature. The fluorescence analyses of the ROH* and RO-* species lead to the detection of three critical concentrations of the surfactants. However, literature describes a wide range of unique CMC values. This contradictory fact can be rationalized by appealing to two different arguments. The first argument relies on the aggregation mechanism. Several models have been proposed for sterol derived surfactants and bile salts where the most widespread are the continuous and the step aggregation processes.31 The second argument relies on the sensitivity of the employed techniques,18 which limits the detection of the critical concentrations. The different fluorescence curves obtained in the present study show a slow change which encompasses, at least, one order of magnitude the surfactant concentration. Using the same fluorescent dye in media containing cationic surfactants of the n-alkyl trimethylammonium family26 (that is, a conventional surfactants), the changes in the fluorescent curves were sharp and they took place at less than one order of magnitude of the surfactant concentration. Thus, the different behavior between the conventional and the sterol derived surfactants should indicate the existence of a progressive aggregation in the case of CHAPSO and CHAPS which 17 ACS Paragon Plus Environment

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includes some critical changes in the structure of the different aggregates. A coherent explanation was suggested by Funasaki et al9 for the case of CHAPS, where the authors classified the degree of aggregation of this surfactant as an intermediate criticalness between sodium taurodeoxycholate (which forms micelles critically) and sodium TC (which self-associates rather noncritically). A similar proposal was presented by Matsuoka et al10 for TC and cholate, deoxycholate and ursocholate.11 They found two CMCs which separated the concentration range into three parts. Below the lower CMC only the existence of monomers was considered, whereas between the two CMCs, the aggregates would grow in size from small and unstable aggregates to big and stable aggregates. Thus, a detailed explanation of the different critical concentrations obtained for CHAPS and CHAPSO was lacking. Our results indicate that below the pre-CMC1, both surfactants are in a monomeric form and when this critical concentration is achieved, the ROH* species is intensely affected by the formation of surfactant aggregates. The pre-CMC1 values obtained for CHAPSO and CHAPS are clearly smaller than the CMCs found in the literature (from 4 mM to 8 mM).9, 20-22, 32, 33 Thus, the pre-CMC1 is indicative of the formation of some kind of small premicellar aggregate. Several authors have proposed the existence of these structures (using either continuous or step models) for bile salts as sodium cholate11,

13, 14, 34

or TC,10, 35 and

even for CHAPS as in the work of Funasaki et al.19 Complementarily, the existence of pre-micellar aggregates of surfactants has been also supported by simulation studies.36, 37

In most of the previously exposed works, the established mechanism of formation of

pre-micellar aggregates involves the so-called back-to-back association, a phenomenon driven by the interaction between the hydrophobic faces of the steroid nuclei of two molecules. This primary interaction implies the orientation towards the bulk of the sterol faces containing the hydroxyl groups, a molecular arrangement compatible with


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the blue shift of the ROH* species in the pre-micellar range observed in our work. The interaction of the protonated photoacid with the pre-micellar aggregates causes a modification of the hydrogen bonds previously established between the water and the dye, which now is located in a less polar environment. This phenomenon reverts the prior excited-state stabilization caused by the accepting water, which was higher than that produced in the ground state29 (this is why, in water, photoacids experience a red shift). There is a decrease of the energetic difference between both states which becomes larger as the hydrogen bond strength increases. Thus, when the dye interacts with the pre-micellar aggregates instead of water, there is an increment in the energetic difference between the ground and the ROH* excited state, which is detected by both the blue shift of the main peak (Figure 3b) and by the emergence of a new component band at 421 nm (Figure 4 and SI3). After the slow growth of the small and unstable aggregates, the CMC1 is detected as a break point in the trend of the RO-* fluorescence. The obtained CMC1 values for CHAPS and CHAPSO are comparable with the standard CMCs described in the literature. The formation of stable micelles involves the arrangement of a less polar environment for the dye and, as can be seen in our results, this fact has an important repercussion on RO-*. In these circumstances, the less charged excited form, ROH*, is favored and a displacement of the equilibrium between both species is observed (Figure 6), that is, the RO-* curve shows a positive slope. In this situation, the dye is confined among the polar faces (but not as polar as the bulk) of the rings, and the blue shift of the ROH* form, which started with the interaction with the pre-micellar aggregates, still keeps on. Concerning the solvatochromism of the RO-* species, it is known that deprotonated photoacids establish hydrogen bonds with donating molecules29 and, thus, the ground-state undergoes a higher stabilization than that of the excited state (a blue shift). In a less polar environment the formation of


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hydrogen bonds between the deprotonated photoacid and the donating solvent is prevented. This causes a decrease in the energy between the excited and ground state, and this is indeed what we observed, a red shift of the RO-* peak (Figure 3b). The third critical concentration, CMC2, was established by taking into account the changes in the ROH* and HPTS* (total) fluorescence (SI5), being the former measurements more accurate. The obtained values were higher than the standard CMCs of the surfactants and, therefore, they should indicate changes in the micellar structure. To our knowledge, there is little literature describing this critical point. Using NMR spectroscopy Qin et al21 also found an unexpected critical concentration for CHAPS, higher than the standard CMC. It was attributed to a change in the micellar structure, affecting particularly the tail protons and suggesting the formation of a new layer of surfactant around the primary micelle. The value they found (32.3 mM) is about three times the one found in our work, but the coincidence in the detection of significant changes in the micelles by two independent methods leads us to consider that they are not an artifact. Origin of the interaction of HPTS with CHAPSO and CHAPS The effect of different parts of the structure of CHAPSO and CHAPS on HPTS was determined using different analogues. CAPS and HAPS were used as analogues of the end of the tails (that is, the head) of the surfactants, and TC as an analogue for the sterol ring and the amide bond of the tail. No modification of the spectral properties of the dye was detected in the presence of CAPS and HAPS (SI 3 to 6). Thus, the interaction of HPTS with the hydroxyl group or the quaternary amine of the surfactants is discarded. Surprisingly, despite the negative charge of the molecule, a clear effect of TC on HPTS was observed (Figures 7 and 8, and SI5 to SI8). This effect was not observed using conventional negative surfactants as sodium dodecyl sulfate and sodium 1dodecanesulphonate.26 In these cases, the high electrostatic repulsion between the 20 ACS Paragon Plus Environment

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charged head of the alkyl surfactants and HPTS totally hampers the association of the dye with the surfactant aggregates. Consequently, the very nature of the interaction between TC and HPTS should rely on the role of the sterol ring and/or the amide bond. At this point, it is interesting to mention that the three critical concentrations found in the case of TC (SI4) support the idea of the existence of the same stages as those found for CHAPSO and CHAPS. Megyesi et al17 also found three different breakpoints (2, 10 and 23 mM) in the evolution of cholate, a similar bile salt. In our case, the pre-micellar concentration observed is compatible with the hypothesis reported by several authors that propose the formation of TC dimers or small aggregates.10, 14, 16 In fact, our preCMC1 (2.72 mM) is coherent with the smaller CMC range (3-5 mM) shown by other authors.14, 16 Complementarily, CMC1 (8 mM) is compatible with the values of Meyerhoffer (8-10 mM) and Funasaki (6.5-8.3 mM), and CMC2 (15 mM) well matches the CMC proposed by Matsuoka (15 mM). This scenario of partial coincidences can be explained by the slow growth of the TC aggregates during their evolution from dimers to stable micelles, where critical points are included and can lead to uncertainty in the determination of the corresponding critical concentrations. As in the case of the interaction between the surfactants and the dye, relevant information is provided by the infrared spectra. The most intense variations in intensity and/or shape corresponded to the peaks assigned to hydroxyl and sulfonate groups (Figures 9 and 10). This fact supports the hypothesis of an interaction between the hydroxyl groups of the ring polar face of CHAPSO, CHAPS and TC, and the sulfonate groups of HPTS. This is also compatible with the fluorescence results of the present work and the back-to-back association of the dimers. Note that in the region of the amide II band (1550 cm-1) the changes are due to the HPTS peaks assigned to aromatic ring deformations30 and, thus, the interaction of the dye with the amine group (or its 21 ACS Paragon Plus Environment

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resonant form) has to be discarded. This is especially relevant because literature describes several cases of interaction with different types of charged amine groups with the sulfonate groups of pyranine.38-41 Consequently, the main driving force for the interaction between the sterol surfactants and HPTS has to be assigned to the formation of hydrogen bonds between the sterol hydroxyl groups and the sulfonates of HPTS. Biological implications The particular evolution of bile salt aggregates and bile salt derivatives and how they interact with several dyes, has led several authors to propose the existence of two different sites where the drugs can be hosted. They confirmed the hosting of apolar molecules in hydrophobic sites of primary aggregates and micelles of these surfactants.12, 23, 25, 42 In the same way, the existence of polar sites is also assumed as a result of the incorporation of molecules containing polar groups into secondary aggregates and into micelles. It is assumed that this incorporation takes place in the regions containing hydroxyl groups, and the characteristics of such regions are modulated by the number of hydroxyl groups.24 In the present work, the results show that CHAPSO, CHAPS and TC are able to host HPTS and that in such as situation the dye is preserved from the bulk medium (inset Figure 6). Thus, their micelles are able to carry not only apolar and polar substances, but also a highly charged negative molecule (HPTS). It is known that the physiological functions of bile salts are related to the emulsification and transport of fats and fat-soluble molecules,43 and several authors have proposed the use of biological and synthetic surfactants for the delivery of amphiphilic and hydrophobic drugs.44 Furthermore, there are simulation studies which support the idea that the particular features of the bile salt micelles facilitate nutrient release to be absorbed in the intestines.45 Thus, taking into account our results, bile salts and some of their derivatives, as CHAPSO and CHAPS, become also candidates to 22 ACS Paragon Plus Environment

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transport and deliver charged molecules. And, surprisingly, this functionality is possible even if the molecules bear negative charges, bypassing the electrostatic repulsion with the negative bile salts. This possibility can also take place at sub-micellar concentrations because, as it has been shown, HPTS notably interacts with the premicellar aggregates of the surfactants (inset Figure 6). This behavior is clearly different from that shown by other polar drugs with cholate,13 as they are incorporated into bile salt aggregates only when secondary aggregates are formed. These results are a novelty which opens the door for potential applications of steroidal surfactants. In this way, further studies have to be done, for example, to deeply characterize the hosting behavior of such molecules in the three detected stages.

CONCLUSIONS HPTS interacts with different aggregates formed by CHAPSO and CHAPS and also with those of the negative bile salt TC. From the changes of the excited forms of the dye, we reveal a broad range of surfactant concentration for the transition from the monomeric form to the micellar state. During this slow evolution, three critical concentrations are established and, thus, four different ranges are considered. In the first, the monomeric form of the surfactants is the only species in the bulk. During the second, the formation of pre-micellar aggregates takes place. The third range involves the formation of micelles. And, in the fourth range, a change in the micellar structure is proposed. The interaction of the dye and the surfactants is driven by the formation of hydrogen bonds between the hydroxyl groups of the sterol ring of the surfactants and the sulfonate groups of the dye. A hosting effect not only of the zwitterionic surfactants, but also of the negative bile salt is documented. This indicates that a highly negative charged molecule can become a guest of the aggregates formed by those substances. Acknowledgements


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We want to thank Joan Suades and Álex Perávalez for their kind gift of HAPS and CHAPS respectively, and to Sándalo Roldán-Vargas for his useful comments. Supporting Information Available Additional UV-visible, fluorescence, and critical concentrations data. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES (1) Odahara, T. Stability and solubility of integral membrane proteins from photosynthetic bacteria solubilized in different detergents. Biochim. Biophys. Acta 2004,

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