Self Assembly of pH-Sensitive Cationic Lysine ... - ACS Publications

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Self Assembly of pH-Sensitive Cationic Lysine Based Surfactants Amalia Mezei, Lourdes Pérez,* Aurora Pinazo, Francesc Comelles, Maria Rosa Infante, and Ramon Pons Departament de Tecnologia Química i de Tensioactius, Institut de Química Avançada de Catalunya, IQAC−CSIC, c/Jordi Girona 18-26, 08034 Barcelona, Spain S Supporting Information *

ABSTRACT: Three cationic surfactants of the type Nε-acyl lysine methyl ester hydrochloride have been studied with respect to solution behavior and adsorption on the air/water interface, as well as the thermolyotropic behavior. The selfassembly of these surfactants, which have the cationic charge on amine protonated groups, was assessed by different physicochemical methods. Depending on the pH value, these surfactants can dissociate in aqueous solutions, losing the cationic charge. Therefore, knowledge of the pKa of these compounds is essential to explain their behavior in aqueous solutions. The bulk techniques, conductivity, and nuclear magnetic resonance diffusion (NMR) obtained similar critical micellar concentration (CMC) values, which were well above those obtained from surface tension. Surface tension measurements were strongly dependent on the technique used, namely, Wilhelmy plate and pendant drop. The phase behavior at medium to high concentrations has been studied by optical polarizing microscopy and small angle x-ray scattering (SAXS). The X-ray studies showed that the lysine-based surfactants at low hydration have rich thermotropic liquid crystalline behavior. The results are discussed in terms of the structure of the compounds and the cationic charge of the molecule. We will show how apparently small changes in molecule structure have a large influence on phase behavior.

1. INTRODUCTION Recently, it has been shown that cationic amphiphiles have great potential in biomedical applications as antimicrobial and antifungal agents in human infections. They can also be used in gene therapy (for instance, cationic vesicles made from cationic surfactants, gemini surfactants, in particular, can encapsulate RNA or DNA for cellular transfer1−3), as vehicles for certain drugs,4,5 and as modifiers of the physicochemical and biological properties of biomaterials (prosthesis and synthetic veins).6 Particularly interesting in pharmacology and medicine are the synthesis and characterization of biodegradable cationic surfactants with low cytotoxicity. Cationic surfactants with these properties can be obtained by hydrophobic modification of cation bearing natural amino acids. Often, these molecules combine charged or noncharged residues [i.e., glutamic acid (Glu), lysine (Lys), arginine (Arg), serine (Ser), leucine (Leu); phenylalanine (Phe), alanine (Ala)] as the hydrophilic headgroup with a hydrophobic tail of different structure, length, and number [i.e., fatty acids, fatty alcohols, fatty amines] as synthons for the amphiphilic structure.7−10 Among the natural amino acids, lysine is an essential low-cost amino acid with two amino and one carboxylic group. The presence of these amino and carboxylic groups makes possible the synthesis of anionic, cationic, and amphoteric surfactants by introducing hydrophobic groups in the carboxylic or amino groups.11 Because of that, this amino acid constitutes a prime raw material for the preparation of biodegradable and biocompatible cationic surfactants. In this regard, our group has recently synthesized cationic surfactants of the type Nε-acyl lysine methyl ester hydro© 2012 American Chemical Society

chloride salts in which the alkyl chain length has been varied from 12 to 16 carbons atoms (Figure 1).12 These lysine

Figure 1. Chemical structure of Nε-acyl lysine methyl ester hydrochloride salt surfactants; n = 10 (LKM), n = 12 (MKM), n = 14 (PKM).

surfactants exhibit antimicrobial properties with minimum inhibitory concentration values in the range 4−125 μg/mL. According to their biodegradation behavior, they can be considered readily biodegradable compounds resulting in lower aquatic toxicity than that of conventional monoquats. Moreover, these cationic surfactants show very low hemolytic character. Given their simple structure and their interesting Received: July 5, 2012 Revised: November 14, 2012 Published: November 19, 2012 16761

dx.doi.org/10.1021/la304117f | Langmuir 2012, 28, 16761−16771

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Figure 2. Dissociation states occurring in the lysine based surfactants in aqueous solutions. The saturation adsorption values (Γ) at the air/water interface were calculated fitting the experimental points before the break to the Gibbs adsorption equation (eq 1)

biological properties, these surfactants can be good candidates in biomedical applications. In this work, we examine the solution-phase behavior of the Nε-acyl lysine methyl ester surfactants for elucidating the type of structures that these compounds form in aqueous solutions in order to establish their application in different technologies. For this purpose, the micelle formation and surface activity in aqueous diluted solutions has been investigated using conductivity, proton activity, NMR self-diffusion, and surface tension. The structures formed at high concentrations have been investigated using SAXS. We will show how the hydrophobic length modulates the self-assembly of these surfactants and how this self-assembly modulates the molecular acidity at low concentrations. The high concentration behavior is governed by the balance between the hydrophobic length and the polar head and by the tendency to form strong hydrogen bonding.

Γ = − 1/nRT dγ /d ln C

(1)

where R is the gas constant, T absolute temperature, γ surface tension, C concentration, and n the number of free species per surfactant molecule. 2.3.2. Pendant Drop Shape Analysis. The surface tension was measured using a homemade pendant drop tensiometer.14,15 In this technique, a surfactant solution drop was created at the end of a straight-cut Teflon tube, which had an internal diameter of 0.8 mm and an external diameter of 1.58 mm. The image of the drop was recorded using a web cam (640 × 480 pixels) and corrected for spherical aberration. The drop profile was then extracted from the corrected drop image after background subtraction. The droplet contour was taken at the point of maximum slope of the intensity. This contour was fitted to the Laplace-Young equation using a homemade golden section search algorithm.16 The input parameters of this algorithm were reference framework, an angular correction for the vertical alignment, the radius of the droplet, and the interfacial tension. Care was taken to ensure a saturated humidity atmosphere to prevent evaporation. Temperature was maintained at 25.0 ± 0.5 °C and also at 40.0 ± 0.5 °C. Pure water surface tension measurements were found in a range of 70 ± 2.0 mN m−1. Further checking of the setup with ethanol found values of 21.2 ± 1.0 mN m−1, both standards agreeing with published values of 72.0 and 21.9 mN m−1 (at 30 °C).17 2.4. Conductimetry. Conductivity was measured using an Orion Cond. Cell 011010A with platinized platinum electrodes in conjunction with a Thermo Orion 550A with a cell constant of 0.998 cm−1. The cell constant was calibrated with NaCl solutions of known conductivities. The conductivity of water was subtracted from the measured conductivity of each sample. Measurements were made at increasing concentrations to minimize errors from possible contamination from the electrode. The measurements were performed at 25 ± 0.1 °C and 50 ± 0.2 °C. 2.5. NMR Measurements. The pulsed-gradient stimulated echo experiment was used to determine the self-diffusion coefficients of the surfactants at 25.0 ± 0.5 °C and 50 ± 0.5 °C. 1H NMR measurements were obtained on a Varian Inova 500 MHz spectrometer equipped with a standard 5-mm indirect-detection, pulsed-field-gradient (PFG) probe. The combination provided a z gradient strength (g) of up to 0.33 Tm−1 (33 Gcm−1). The values of CMC were determined using the two-site exchange model.18,33 2.6. Optical Polarizing Microscopy. An Olympus BX51 polarized light microscope equipped with a Linkam TMHS 600 hot stage, controlled by TP94 unit, was used. The temperature control allows a ±0.1 °C resolution. Images were acquired with an Olympus C-5060 Wide Zoom digital camera. Anisotropic liquid-crystalline phases give rise to typical birefringent textures under polarized light. 2.7. Small-Angle X-ray Scattering (SAXS). SAXS measurements were carried out by using a Kratky Camera of small angle (M-Braun)

2. MATERIALS AND METHODS 2.1. Materials. NaOH and Borax were purchased from Sigma Aldrich and HCl and Brij35 from Merck. The acyl lysine methyl ester surfactants were obtained with a purity of 99% by a three-step procedure: (a) methylation of the acid group of the commercial Zlysine, (b) condensation of the acyl chloride to the ε-amino group of the Z-lysine methyl ester, and (c) removal of the Z protecting group by hydrogenation. More details on the synthesis are reported in the literature.13 The studied surfactants were the Nε-lauroyl lysine methyl ester (LKM), Nε-myristoyl-lysine methyl ester (MKM), and the Nεpalmitoyl-lysine methyl ester (PKM). Millipore water from a milli-Q four-bowl system was used in preparing all sample solutions. 2.2. Proton Activity. 2.2.1. pKa Measurements. The pKa values were determined from the potentiometric titration of 1 mL (for LKM) or 1.5 mL (for MKM and PKM) of 5 mM aqueous surfactant solutions with 5 mM NaOH aqueous solutions. The pH electrode was an ORION 8103SC semimicro and the potentiometer was a Thermo Orion model 720A+. All titrations were conducted at 25 ± 0.1 °C and under nitrogen gas atmosphere and magnetic stirring. pKa was determined as the pH of the corresponding semi equivalence point. 2.2.2. pH-Surfactant Concentration Measurements. The pH values of different concentrations of surfactant water solutions under nitrogen were measured using a pH electrode (model ORION 8103SC semimicro). Measurements were made at increasing concentrations of surfactant to minimize errors from possible contamination from the electrode. 2.3. Surface Tension Measurements. 2.3.1. Wilhelmy Plate. Surface tension measurements were performed at 25 ± 1 °C using a Wilhelmy plate coupled to a Krüss K-12 tensiometer. 16762


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Figure 3. Titration curve of 5 mM aqueous solutions of LKM (solid squares), MKM (solid circles) at 25 °C, MKM (open circles) at 50 °C, and PKM (solid triangles) at 50 °C in the absence of additives, MKM in the presence of 40 mM Brij35 micelles (open triangles) at 25 °C. and a Siemens KF 760 (3KW) generator. The wavelength corresponding to the Cu Kα-line (1.542 Å) was used. The linear detector was PSD-OED 50 M-Braun, and the temperature controller was a Peltier KPR AP PAAR model working with ±0.1 °C resolution. The samples were measured either in flame-sealed glass capillaries or using a paste sample holder closed by Mylar windows. Because of using a line collimated beam, our scattering curves show mainly slitlength smearing. The SAXS scattering curves are shown as a function of the scattering vector modulus

q=

4π sin θ /2 λ

The obtained pKa values indicate that these surfactants have weak acidic properties; consequently, the cationic character will depend on the pH of the medium. This behavior is common in amino acid based surfactants as well as in other cationic surfactants in which the cationic charge is on a protonated amine group.19−24 Taking into account that the pKa of the αamino group of lysine is 8.7, we can state that the introduction of hydrophobic groups in the amino acid drastically reduce the apparent pKa and the decrease is higher as the hydrophobic group becomes longer. This effect is an induced effect due to aggregation, not directly attributable to chemical bonding. The initial pH of the solutions is higher for LKM than for MKM, which is slightly higher than for PKM. In the former case, the concentration is around the CMC (see below for more details about the CMC), while the other two are well above the critical aggregation concentration. Therefore, without adding NaOH, the acidic character increases with the chain length of the surfactant via the effect on the CMC. The apparent pKa shift of molecules undergoing aggregation has been extensively studied before,21−24 and two main contributions to the pKa shift have been identified. One contribution comes from the virtual charge effect due to a discontinuity in dielectric constant at the micellar surface24 and a second contribution comes from the polar head interaction. We can attribute the difference from the pKa of the α-amino group of lysine to the apparent pKa of LKM to the presence of both effects. The further reduction of apparent pKa for MKM and PKM (0.9 and 1.7 pKa units, respectively) could be attributed to the contribution of changes in the shape of the aggregates. In order to discard the effect of temperature as the origin of this difference, we note that the difference between pKa values of MKM and PKM at 50 °C is 1.0. The influence of the shape of aggregates has already been suggested as the origin of the differences found for the values of the interaction parameters in the literature as a function of concentration and chain length.21,25 The differences encountered here are bigger than the differences found by Maeda between dodecyl and tetradecyl amine oxide surfactants (0.4 pKa units). As a complementary experiment, we titrated the MKM compound in the presence of a large amount of Brij35. Brij35 is known to form spherical micelles with little dependence of size on concentration.26 By diluting MKM, we expect two effects: on one hand, the head−

(2)

where q is the scattering angle and λ the wavelength of the radiation. The q range obtained with our setup was from 0.2 to 6 nm−1.

3. RESULTS AND DISCUSSION 3.1. Proton Activity. The Nε-acyl-lysine compounds studied in this work (Figure 1) have the cationic charge on the protonated α-amino group of the amino acid. One practical method to study these products is the determination of their protonation constant by potentiometric titration. These surfactants present an acid−base equilibrium in aqueous solutions. The dissociation states of these surfactants are shown in Figure 2. Lysine-based surfactants can lose the cationic charge depending on the pH of the medium. The solubility of the surfactants was assessed in order to determine if the Krafft temperature was above or below room temperature. Krafft temperature was determined with conductivity measurements. (Supporting Information Figure S1). The Krafft temperatures for LKM and MKM are below room temperature (16.5 and 18.8 °C), and for PKM, it is 28.2 °C. This value indicates that the solubility of these surfactants decreases as the length of the hydrophobic chain increases, as it could be expected. The apparent pKa values have been determined from the pH value at the point of semiequivalence in the titration curves shown in Figure 3. These values, also shown in the Figure 3, are the following: LKM = 6.2, MKM = 5.3 (5.5 at 50 °C), and PKM = 4.5. The surfactant concentration of the solutions at the equivalent points is above or close to the critical micellar concentration (CMC) for all of them (see below surface tension results). 16763


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Figure 4. pH values against surfactant concentration for the LKM (circles), MKM (squares), and PKM (triangles) surfactants. PKM measured at 50 °C.

Table 1. CMC Values of LKM, MKM, and PKMa surface tension LKM MKM PKM

conductivity

pH

NMR

CMCWilhelmy plate (mM)

CMCpendant drop (mM)

CMC (mM)

CMC (mM)

CMC (mM)

0.44 ± 0.03 0.023 ± 0.003 0.0027 ± 0.0005

1.8 ± 0.2 0.088 ± 0.010 0.13 ± 0.05b

8.5 ± 1 3±1 0.65 ± 0.1

0.8 ± 0.2 0.09 ± 0.1 0.009 ± 0.002

5.5 ± 1 1.6 ± 0.5 0.6 ± 0.2

Data obtained at 25 °C except conductivity, pH, and NMR for PKM, which are at 50 °C. bThe measurement carried out at 40 °C gave a value of 0.055 ± 0.006 mM.

a

lysine; the process of micellization (in the area of milimolar concentrations) is apparent as a gradual decrease of pKa up to the formation of micelles and the apparent pKa stabilizes at a value slightly below 6, which compares well with the pKa obtained from titration at concentrations around the CMC and seems to imply a minor effect of the ionic strength on the values obtained from titration at these concentrations. For MKM, the pKa stabilizes at 5.3 around 3 mM, while for PKM, the pKa goes through a shallow minimum with a pKa of 3.5 around 0.6 mM and increasing to 3.7 at 1 mM. A reduction of the apparent pKa implies a decrease of the stability of the nondissociated species (we have to keep in mind that for the hydrochloride the nondissociated species correspond to the cationic species). Finally, we have to stress that the pKa values obtained from these measurements closely coincide with the titration values, although both are obtained in different ionic strength conditions. For these pH-sensitive surfactants, pH measurements as a function of surfactant concentration also provide a simple method for the determination of the CMC. Referring to the studied surfactants, we can observe that at low and high concentrations the pH values follow the expected slope for weak acids, with pKa corresponding to the monomers at low concentrations and to the micelles at high concentrations. The onset of micellization will be marked by the first change of slope (at the formation of micelles, a release of protons is produced due to the pKa shift of the micelle with respect to the monomer). Between these two slopes, we can observe an intermediate break, which should be due to the structural

head interaction will be reduced, and on the other hand, the form of the micelles will remain spherical due to the high proportion of Brij35 (8:1). The observed pKa 6.8 is slightly higher than that of LKM. By considering the different contributions, it is reasonable to attribute this latter pKa shift (from the 8.7 value of lysine to 6.8) to the virtual charge effect.24 The remaining difference between 6.8 and 6.2 for LKM should be attributed to the polar head interaction, while the bigger pKa shifts of pure MKM and PKM are attributed to the form and size changes in aggregation.25 This behavior could be interesting in medicinal chemistry, because changes in the protonation state of pH-titrable headgroups would lead to changes in headgroup area and, as a result, in their aggregation state. Surfactants with this type of behavior have been shown to be efficient vectors for gene therapy because the release of DNA into the cells is improved and, consequently, the level of gene transfection may be increased.27 In Figure 4, the pH as a function of surfactant concentration has been plotted for LKM, MKM, and PKM. In the same figure, the expected behavior for weak acids (nonaggregating) of different pKa values is also shown. All surfactants produce a considerably higher concentration of protons than expected from the Nα-lysine pKa (8.7). For the studied surfactants, at low concentrations the apparent pKa is smaller (7.4) than for the free lysine, which could be attributed to the presence of long hydrophobic chains. In all cases, the apparent pKa decreases as the surfactant concentration increases. For LKM, the apparent pKa before micellization is around 1 unit below that of Nα16764


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Figure 5. Conductivity as a function of concentration for LKM, MKM, and PKM. Experimental points and contributions of the separate ions are shown as protons (dashed lines), chloride (dotted lines), surfactant ion (dash-dot lines). The full lines correspond to the pseudophase model calculated as explained in the text. Measurements obtained at 25 °C for LKM and MKM and at 50 °C for PKM.

changes of the formed aggregates, in the case of LKM in oblates and in the case of MKM and PKM probably in lamellae. Table 1 lists the values of critical micellar concentration determined by different methods. The most remarkable of these results is the low CMC values obtained by the Wilhelmyplate method (see later discussion) as compared to the values obtained from pendant drop and the bulk methods. Figure 5 shows the change of conductivity (λ) for aqueous solutions of the cationic surfactants as a function of concentration. The conductivity values fit into two straight lines of different slopes; the CMC corresponds to the abrupt change of the slopes.28 The obtained CMC values are on the same order as those determined by NMR measurements (see Table 1 and Figure 5S). A simple pseudophase model for calculating the conductivity is shown as a full line in Figure 5. Also, the separate contributions of proton, chloride, and surfactant cations are shown for comparison in the same figures. The proton contribution was obtained from the pH value and the single ion equivalent conductivity from the literature.17 Chloride concentration was taken equal to the total surfactant concentration below the CMC. The surfactant ion concentration was calculated taking into account the acid−base equilibrium and effective pH, and we used an equivalent conductivity of 20 in consistent units to calculate this ion

contribution. The activity coefficients were taken into account using Davies equation.29 The pseudophase model conductivity has been calculated using single ion contributions before the CMC and fractional contribution of the chloride and surfactant cation above the CMC. This simple model gives values around 0.7 for the counterion binding, which are reasonable. The contribution of the different single ions to the global conductivity of these surfactants shows clearly that the acid− base equilibrium has a strong influence on PKM, while it is only marginal for MKM and LKM. 3.2. Surface Tension. The adsorption of these surfactants has been investigated by surface tension measurements. Figure 6 shows the surface tension−concentration curves obtained for the three surfactants using two different techniques, the Wilhelmy-plate and the pendant drop shape analysis. The surface tension curves obtained using the Wilhelmyplate (Figure 6A) are characteristic of surfactants; at low surfactant concentrations, high surface tension was measured. With increasing concentrations, surface tension values decrease until a plateau attributed to the formation of micelles is reached. At higher concentrations, the surface tension value is constant. The CMC value is obtained at the slope discontinuity in the surface tension vs log(C) plots. The CMC values (Table 1) of the studied surfactants increase with decreasing length of their hydrophobic chain. However, the CMC values obtained are extremely low, 1 to 2 orders of magnitude lower than those 16765


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CMC values as determined from these two surface techniques and the bulk techniques can be compared. One possible reason for the differences between the two surface methods is that in the case of the Wilhelmy-plate method the wetting properties of the amine groups of the surfactants on the platinum plate play an important role in the measured surface tension. Earlier studies30,31 had shown that the adsorption of amine groups on the platinum surface forms a layer of molecules adsorbed on a surface, which may profoundly alter the wetting properties of the plate. The multilayer formation influences the surface tension measurements increasing the contact angle and thus decreasing the value of the measured surface tension. The smaller CMC of the amino acid based surfactants determined from these measurements could be attributed to the amine group adsorption with the attribution of the faster surface tension reduction. In addition, some discrepancies can arise due to the PKM solubility at room temperature. The PKM shows limited solubility in water at room temperature as compared to LKM and MKM. This could be the reason for somewhat increased scattering of the experimental points obtained at 25 °C from the pendant drop method. With the Wilhelmy plate method, the measurements were done at room temperature, but with the pendant drop method, our setup allowed for measurements at higher temperature. To avoid the solubility/temperature effect for PKM, measurements were done at 40 °C with the pendant drop method, and at this temperature, the surface tension measurements of PKM showed a “normal” behavior. This can be observed in Figure S2 of the Supporting Information where the data at 40 °C is compared to that at 25 °C. In addition, the pendant drop experiments with the longer hydrocarbon chains (MKM, PKM) show bigger scattering, or even some steps, in the decreasing part of the curves. This is more pronounced at longer times (more than 1 h measurements), and in this concentration range, the value of surface tension for repeating measurements is very scattered (time evolution at 25 °C is shown for MKM in Supporting

Figure 6. Surface tension versus surfactant concentration for LKM (□), MKM (○), and PKM (Δ) surfactants. In the first plot are the results from Wilhelmy-plate measurements (A) and in the second one the data from pendant drop measurements (B).

obtained from conductivity or NMR. This prompted us to use an alternative method for surface tension determination. The surface tension as obtained from pendant drop shape analysis is shown in Figure 6B. The shape of the curves is similar to that obtained using the Wilhelmy-plate; however, the stabilization of the surface tension is observed at higher concentrations for each surfactant. This can be clearly seen in Table 1 where the

Figure 7. Surface tension as a function of surfactant concentration at different pH values. Measurements were done at 25 °C. The solutions were buffered at pH 9 using 20 mM borax and at pH 2 using 10 mM HCl. 16766


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Table 2. Micellization Parameters of LKM, MKM, and PKM for the two Surface Tension Methods at 25 °C γCMC (mN m−1) LKM MKM PKM

Γmax × 10−10 (mol/cm2)

pC20

Wilhelmy plate

pendant drop

Wilhelmy plate

pendant drop

Wilhelmy plate

pendant drop

30.0 ± 1.0 37.4 ± 1.0 41.5 ± 1.0

36.7 ± 1.0 34.4 ± 2.0 35.5 ± 1.0

3.88 ± 0.03 5.54 ± 0.05 6.15 ± 0.07

3.78 ± 0.05 4.82 ± 0.05 4.61 ± 0.06

8.5 ± 2.0 2.8 ± 0.7 3.4 ± 0.9

2.7 ± 1.5 5.5 ± 3.0 4.2 ± 2.0

It is also observed that pC20 increased slightly by an increase in the length of the hydrocarbon chain in ionic surfactants. The high pC20 value indicates that the effectiveness of these surfactants in reducing the surface tension at the air/water interface is extremely high, similar to that shown by Gemini surfactants.34 The calculated Γmax values change with the alkyl chain length from 12 to 16 carbon atoms. Due to uncertainties in the values, large errors in Γmax were obtained. The large values for Γmax suggest a closely packed hydrocarbon group (see X-ray measurements below) and relatively large pC20. Unexpected values have also been obtained for the area per molecule (Amin; see Supporting Information Table S1). The uncertainty on the surface tension−concentration plots is large, and the corresponding unpredictability of the minimum area per molecule is also large. This dilemma together with the possible changes in the value of n makes it very difficult to rationalize the values obtained. However, the n = 1 is also favored by fitting the oblate core−shell model to experimental SAXS on LKM (see Supporting Information Figure S4). The calculated area per molecule is close to the value found from the pendant drop (0.70 nm2 compared to 0.62 nm2) confirming that at natural pH the number of adsorbed species is 1 rather than 2. The spectra of MKM, also shown in Figure S4, show a more peak-like behavior at intermediate scattering vectors and a decidedly stronger intensity at small q. This corresponds to the formation of bigger aggregates; in this case, the fitting corresponds to that of multilamellar structures. The calculated area per molecule for MKM is 0.79 nm2. The most striking feature of the obtained values is the great difference between the values by surface tension and the other techniques. The CMC values obtained by conductivity and NMR are in reasonable agreement with the results reported for cationic surfactants with similar alkyl chain length.34−37 The disagreement between surface tension and bulk techniques in the determination of the critical micellar concentration or critical aggregation concentration is present in the literature16,38,39 The discrepancy can be explained by the different sensitivity of the bulk methods as compared with the surface methods. At very low concentration, the water dissociation is significant compared to the surfactant concentration, and a significant amount of unprotonated species is produced (pH around 5). Those unprotonated species are neutral and, therefore, have a lower solubility than the charged species. The neutral species adsorbs at the interface and can be responsible for the reduction of surface tension at low surfactant concentration. When increasing the concentration, according to ionic equilibrium the proportion of neutral species decreases but its total concentration does not. It was observed that, at high concentration, after one hour, the solutions showed some turbidity that can be a sign of the formation of bigger aggregates. This should be due to structural changes of oblate micelles growing and forming lamellae. At higher concentration, the pH of the aqueous solution decreases down to values about 3.5, and the protonated species

Information Figure S3). This could be a consequence of some time evolution due to precipitation. The surface tension of these surfactants is also influenced by the pH medium: the smaller the pH of the solutions, the bigger the CMC. The effect of pH is shown for MKM in Figure 7. The small differences found as a function of pH may be a consequence of the importance of the hydrogen bonds of the polar head, which minimizes the effect of the ionic charge. It is also noteworthy that the difference between the measurements at natural pH (∼4) and pH 9 is smaller than the measurement at pH 2. This seems to show that at natural pH most of the adsorption effect can be attributed to the neutral species (deprotonated amine), while only at very low pH does most of the surfactant retain its charge. The evaluation of the area per molecule, using eq 1, shows that at pH 9 and natural pH the areas are close to each other (using n = 1 as the number of adsorbing species), while at pH 2, the use of 2 as the number of adsorbing species results in nearly constant area per molecule (0.30 compared to 0.37). Complementary electrophoretic mobility experiments showed that at pH 2 the ζ-potential was 44 mV while at natural pH it was 25 mV and at pH 13 it was −32 mV (at this latest pH, the methyl ester should be partially or completely saponified). On the other hand, according to X-ray data (as shown below), a closer packing of monomers occurs in the case of PKM. As observed previously by Perez et al.,12 a correlation of CMC value with micelle shapes is observed. From diffusion NMR studies, it was found that the LKM derivative formed spherical micelles while the MKM formed elongated micelles, and the PKM derivative had low solubility at 25 °C but formed micelles at 50 °C. Cationic surfactants with the same alkyl chain have CMC values that are similar (considering the bulk methods) or 1 order of magnitude higher (considering the pH and surface tension methods). For instance, the dodecyl trimethyl ammonium bromide CMC is 10 mM, while that of the Nαdodecyl arginine methyl ester hydrochloride is 4−6 mM depending on the technique.32 The values of surface tension at the CMC, the pC2034 (minus logarithm of the surfactant concentration needed to reduce the surface tension of water by 20 mN m−1), and the maximum surface densities calculated using the Gibbs adsorption isotherm with (eq 1) are presented in Table 2. The calculations in this case were made by n = 1, as a correct estimation of the number of adsorbing species is problematic. The effectiveness of surface tension reduction (γCMC), according to pedant drop method, has no clear trend as a function of the hydrocarbon chain length. However, by Wilhelmy-plate method the effectiveness is greatly increased by the decrease of the alkyl chain length, with LKM being a very effective surface tension reducer (see Table 2). This observation agrees with the earlier results of Rosen et al.,33 which were found for cationic surfactants in 0.1 M NaCl solution (γCMC change between 31.5 and 38 mN m−1 for Ntrimethylated surfactants with C12−C16 carbon atoms). 16767


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Figure 8. SAXS curves for the three lysine-based surfactants at 50 °C.

In Figure 9, an example of the complex thermolyotropic phase behavior of the single chain amino acid compounds is

dominated the formation of the classical micelles in the bulk solution that can be observed by NMR (Supporting Information Figure S5) and conductivity. In this case, the decrease of the CMC value with the enlargement of the alkyl chain agrees with the performance of ionic surfactants. Analogous behavior has been reported for arginine-based diacyl surfactants. Using a combination of surface tension, pH, and LS measurements it was observed that, at very low concentrations, the adsorption of nonprotonated species dominated, while at higher concentrations, the protonated species dominated the formation of micelles.45 A similar non-ionic-like dependence on chain length has already been reported for alkyl 2-amino-2deoxy-β-D-glucopyranosides46 and lactylamines;47 both types of surfactants have one amine group on the polar head that can be protonated or not, depending on pH values. 3.3. Aggregation Properties at High Concentrations. The thermolyotropic behavior of amphiphiles is of fundamental importance, since it allows the establishment of structure/ behavior/performance relationships.48,49 In this work, the thermolyotropic behavior of the three lysine-based surfactants has been analyzed using SAXS. The X-ray studies show that the lysine-based surfactants at low hydration have rich thermotropic liquid crystalline behavior. All three compounds crystallize as lamellar phases in the dry state. No special precaution was taken to dry the samples further than routine freeze−drying procedures and under-lamp sample loading to avoid atmospheric moisture. In Figure 8, the SAXS curves for the three surfactants are shown at 50 °C in the dry state. For the three products, two or three orders of repetition peaks can be observed, with q positions corresponding to 1:2:3 spacing, characteristic of lamellar structures. These lamellar arrangements correspond to interdigitated hydrophobic chains, in which each hydrophobic chain interpenetrated with the hydrophobic tail of molecules corresponding to another leaflet, with lamellar thickness increasing with hydrophobic chain length. This structure is preserved up to the melting point. The bilayer thickness corresponds to 2.83, 3.08, and 3.24 nm for chains with 12, 14, and 16 carbon atoms, respectively, while the area per molecule remains constant at 0.44 nm2.

Figure 9. SAXS curves for PKM in the dry state at 25 °C (the bottom thin line) and samples of PKM with 10% water at 25, 45, 60, 70, 80, and 90 °C from bottom to the top.

shown. While in the dry state, PKM shows just a lamellar arrangement up to the melting point, the addition of a small amount of water (10% w/w) already produces dramatic effects on the liquid crystalline structures. At 25 °C, the addition of water produces the presence of a peak to the left of the main peak in the lamellar structuring that still appears in the same place as in the dry product. This peak can be attributed to a non-interdigitated lamellar structure, while that corresponding to the dry state has been assigned to an interdigitated lamellar phase. At this temperature, the system does not seem to accept more water than necessary for expanding the structure to noninterdigitated. Increasing the temperature produces changes that can be attributed to the formation of bicontinuous cubic structures in the presence of the interdigitated lamellar (1q, 2q, 3q). The cubic bicontinuous structures can be assigned to Pn3m (√2, √3, √8 peak positions with the third and fourth peaks scarcely observed), then Ia3d (peak positions (√6, √8), 16768


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Figure 10. Representative optical micrographs in cross-polarized configuration displayed by PKM and PKM/water mixture: (a) dry PKM at 25 °C; (b) PKM/water at 25 °C; (c) PKM/water at 70 °C; and (d) PKM/water at 25 °C after heating to 100 °C and then cooling down. The width of the pictures corresponds to 850 μm.

and finally Pn3m at the higher temperatures.49 The surfactant bilayer arranged in those cubic bicontinuous structures has parameters compatible with the ones calculated for the lamellar non-interdigitated structures found at low temperatures. Parallel observations with optical polarizing microscopy provide complementary information concerning the identification of the phases.50 They are useful as a fast mapping tool to complement the SAXS observations; however, some discrepancies may be present because of different sample preparation and conservation under the measurement conditions (for instance, concerning water evaporation). In Figure 10, we show an example with dry PKM and PKM/water mixtures at different temperatures. At 25 °C, both samples have the same texture. This is quite undefined and may be compatible with the coexistence of a double lamellar structure as deduced by SAXS. With increasing temperature, the texture of dry PKM is the same, agreeing with the invariable structure detected by SAXS. However, for the PKM/water sample starting from 60 °C a mosaic structure with crystals, possibly hexagonal, can be observed. This disagrees with the assignation made by SAXS, which corresponds to a cubic structure (which would be non-birefringent) coexisting with lamellar. The behavior as a function of increasing concentrations also shows complex thermolyotropic capacity, not just for PKM but also for LKM and MKM. This thermolyotropism deserves further investigation, in particular, by complementing the SAXS results with WAXS. Preliminary results with WAXS suggest that with this technique further information concerning the structuring of these mesophases can be obtained. This information may further clarify the role played by the

headgroup interactions (in particular, concerning the multiple hydrogen bond possibilities) and hydrophobic tails.

4. CONCLUSIONS The Nε-acyl lysine derivative surfactants present a peculiar manner of aggregation governed by acid−base processes. From the comparison of Wilhelmy and pendant drop surface tension, a big discrepancy was observed, up to 1 order of magnitude higher CMC values for the pendant drop method; this discrepancy should be attributed to specific binding of ammonium groups to the platinum plates. Difficulties of interpretation have also been encountered due to the limited solubility of PKM at room temperature, measurement at 40 °C using the pendant drop technique results in normal behavior. At concentrations higher than the pendant drop surface tension CMC, the pH of the solutions decreases, and they act as ionic surfactants, forming the classical micelles that can be observed by NMR and conductivity. Also, it has been shown that pKa and pH sensitivity of these surfactants can be tuned by the free amino group of the amino acid, the alkyl chain length, and the type of structure of the surfactant. The behavior of the pH as a function of concentration can be rationalized in terms of micellization plus micellar shape transformation; small and spherical micelles produce moderate changes of apparent pKa, while lamellar structures induce stronger shifts. The surface tension as a function of pH shows that the behavior at natural pH is very close to that observed at pH 9, while only getting to a pH as low as 2, clear differences can be 16769


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and Multi-Amine Headgroups. J. Phys. Chem. B 2008, 112, 1409− 1413. (7) Takehara, M. Properties and applications of amino-acid based surfactants. Colloids Surf. 1989, 38, 149−167. (8) Boyat, C.; Rolland-Fulcrand, V.; Roumestant, M. L.; Viallefont, P.; Martinez, J. Chemo-enzymatic synthesis of new non ionic surfactants from unprotected carbohydrates. Prep. Biochem. Biotechnol. 2000, 30, 281−294. (9) Gerova, M.; Rodrigues, F.; Lamere, J. F.; Dobrev, A.; FeryForgues, S. Self-assembly properties of some chiral N-palmitoyl amino acid surfactants in aqueous solution. J. Colloid Interface Sci. 2008, 319, 526−533. (10) Vijay, R.; Angayarkanny, S.; Bhasker, G. Amphiphilic dodecyl ester derivatives from aromatic amino acids: Significance of chemical architecture in interfacial adsorption characteristics. Colloids Surf., A 2008, 317, 643−649. (11) Colomer, A.; Pinazo, A.; García, M. T.; Mitjans, M.; Vinardell, M. P.; Infante, M. R.; Pérez, L. pH-Sensitive Surfactants from Lysine: Assessment of Their Cytotoxicity and Environmental Behavior. Langmuir 2012, 28, 5900−5912. (12) Pérez, L.; Pinazo, A.; García, M. T.; Lozano, M.; Manresa, A.; Angelet, M.; Vinardell, M. P.; Mitjans, M.; Pons, R.; Infante, M. R. Cationic surfactants from lysine: synthesis, micellization and biological evaluation. Eur. J. Med. Chem. 2009, 44 (5), 1884−1892. (13) Colomer, A.; Pinazo, A.; Mitjans, M.; Vinardell, P.; Manresa, A.; Pérez, L. Cationic Surfactants Derived from Lysine: Effects of Their Structure and Charge Type on Antimicrobial and Hemolytic Activities. J. Med. Chem. 2011, 54, 989−1002. (14) Zuo, Y. Y.; Ding, M.; Li, D.; Neumann, A. W. Further development of Axisymmetric Drop Shape Analysis-Captive Bubble for pulmonary surfactant related studies. Biochim. Biophys. Acta 2004, 1675, 12−20. (15) Anastasiadis, S. H.; Chen, J. K.; Koberstein, J. T.; Siegel, A. F.; Sohn, J. E.; Emesson, J. A. The Determination of Interfacial Tension by Video Image Processing of Pendant Fluid Dropes. J. Colloid Interface Sci. 1987, 119 (1), 55−66. (16) Pinazo, A.; Angelet, M.; Pons, R.; Lozano, M.; Infante, M. R.; Pérez, L. Lysine-Bisglycidol Conjugates as Novel Lysine Cationic Surfactants. Langmuir 2009, 25, 7803−7814. (17) Weast, R. C., Ed. Handbook of Chemistry and Physics, 64th ed., CRC Press Inc: Boca Raton, 1983; pp F33−34. (18) Stilbs, P. Prog. NMR Spectrosc. 1987, 19, 1−45. (19) Boullanger, P.; Chevalier, Y. Surface active properties and micellar aggregation of alkyl 2-amino-2-deoxy-β-D-glucopyranosides. Langmuir 1996, 12, 1771−1776. (20) Tabohashi, T.; Kazuhito, T.; Kazutami, S.; Junichi, K.; Shoko, Y.; Hideki, S.; Masahiko, A. Solution properties of amino acid-type new surfactant. Colloids Surf., B 2001, 20, 79−86. (21) Goldsipe, A.; Blankschtein, D. Titration of Mixed Micelles Containing a pH-Sensitive Surfactant and Conventional (pHInsensitive) Surfactants: A Regular Solution Theory Modeling Approach. Langmuir 2006, 22, 9894−9904. (22) Goldsipe, A.; Blankschtein, D. Molecular-Thermodynamic Theory of Micellization of pH-Sensitive Surfactants. Langmuir 2006, 22, 3547−3559. (23) Hagslatt, H.; Söderman, O.; Jönsson, B.; Lennart, B.; Johansson, A. Divalent Surfactants. General Characterization of the Dodecyl-1,3propylene-Bis(ammonium chloride)/Water System. J. Phys. Chem. 1991, 95 (4), 1703−1710. (24) Söderman, O.; Jönsson, B.; Olofsson, G. Tritation of Fatty Acids Solubilized in Cationic and Anionic Micelles. Calorimetry and Thermodynamic Modeling. J. Phys. Chem. B 2006, 110, 3288−3293. (25) Maeda, H.; Kakehashi, R. Effects of protonation on the thermodynamic properties of alkyl dimethylamine oxides. Adv. Colloid Interface Sci. 2000, 88, 275−293. (26) Basheva, E. S.; Kralchevsky, P. A.; Danov, K. D. The colloid structural forces as a tool for particle characterization and control of dispersion stability. Phys. Chem. Chem. Phys. 2007, 9, 5183−5198.

observed as an increase of CMC. This suggests that at natural pH a significant amount of surfactant is deprotonated. Besides the different lyotropic phases formed by these compounds in water, mainly dictated by the surfactant packing parameter, these molecules can form thermotropic mesophases, some of liquid-crystalline nature, when heated in the absence of solvent, from solid to isotropic liquid phase. The self-assemblies of these molecules are driven by the shape, polarity, charge, and molecular geometry of the molecules. In the dry state, some of the compounds just “crystallize” in lamellar structures, with these being gel or liquid crystals. In the concentrated regime, the cationic surfactants show rich thermolyotropic behavior, as exemplified by the PKM results.

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ASSOCIATED CONTENT

S Supporting Information *

Conductivity as a function of temperature, surface tension− concentration curve for PKM at 40 °C and for MKM by the pendant drop method as a function of time are provided together with minimum area per molecule for all of the surfactants studied as obtained from the Gibbs isotherm, small angle X-ray diffraction for 100 mM LKM and 4 mM MKM aqueous solutions, and diffusion coefficients as a function of concentration for the three surfactants. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Jaume Caelles from the SAXS-WAXS service at IQAC and Imma Carrera are acknowledged for technical assistance. This work was supported by CSIC through a JAE-DOC2010-097 contract cofinanced by FSE 2007-2013, Project CTQ200914151-C02-02, and CTQ2010-14897 from Ministerio de Economi á y Competitividad (Spain). The anonymous reviewers are also acknowledged for comments and suggestions that helped to improve the manuscript.

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REFERENCES

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