Spontaneous Vesicle Based Excipient Formation in Mixtures of

Feb 6, 2015 - Can a catanionic surfactant mixture act as a drug delivery vehicle? Sampad Ghosh , Anirban Ray , Nabakumar Pramanik , Balram Ambade. Com...
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Spontaneous Vesicle Based Excipient Formation in Mixtures of Sodium N‑(n‑Alkanoyl)‑L‑alaninate and N‑Cetylpyridinium Chloride: Effect of Hydrocarbon Chain Length Sampad Ghosh* and Anirban Ray Department of Chemistry, National Institute of Technology, Jamshedpur 831014, India S Supporting Information *

ABSTRACT: The hydrocarbon chain length effects of sodium N-alkanoyl-L-alaninate and N-cetylpyridinium chloride surfactants have been studied at various mixing ratios and concentrations. Both cationic and anionic surfactants form micellar solution in water, whereas by mixing the two surfactants in different compositions and concentrations, stable unilamellar vesicles are formed spontaneously. The variation of the chain length of the anionic surfactant produces a large effect on the stability of mixed surfactant systems. The vesicles formed by the mixtures are found to be thermodynamically stable in different composition and concentration in aqueous media. These vesicles formed by surfactant mixtures are stable for more than one month both for anionic- and cationic-rich side in very dilute solutions. The stability of the vesicle was examined by measuring the turbidity, which remains unchanged for a long time. Dynamic light scattering as well as transmission electron microscopic experiments were performed to investigate the structural properties of the different types of aggregates as well as the formation of vesicular structures. The effect of salt, pH, temperature, and cholesterol on the microviscosity of the domains was evaluated by using steady-state fluorescence depolarization method. Confocal fluorescence microscopic images technique and conductivity measurements have confirmed hollow sphere structure of the vesicles. In the presence of 10 mol % cholesterol and varying solution pH, the mixed surfactant vesicles exhibited leakage of the encapsulated calcein dye, which showed potential application in pH-triggered drug release. The surface chemical properties of the mixed surfactant system, stability, and potential applications of vesicle have been studied systematically.

1. INTRODUCTION The widespread use of surfactant mixtures in industry has stimulated the interest of many researchers. Consequently, in recent years, much work has been done on the solution properties of mixed surfactant systems. However, most of these investigations have dealt with the study of certain physical properties of the solution, for example, the variation of the cmc and the size or micellar aggregation number with the composition of the system.1−5 Recent studies in the interaction of mixed cationic−anionic surfactants also suggest strong synergistic behavior, which is different from individual surfactants.6 Since structural properties of the mixed aggregates can be substantially different from those of pure single surfactant, many recent investigations were concerned with different structural aspects of micelles composed of two different surfactants. It is also widely recognized that a number of micellar properties depend on the structural and dynamic features of the aggregates, which are related to composition of the aggregates. Another interesting aspect that has been less studied is the microenvironmental properties, such as so-called micropolarity and microviscosity, which also depend on the composition and concentration of the mixed micelles.7−9 These are of interest in many technical applications, for example, in micellar catalysis and drug delivery.10−13 Mixture of two oppositely charged single chain surfactants produce a pseudodouble-tailed surfactant through ion-pairing, which is commonly referred to as “catanionic” surfactant.6 Owing to a cylindrical shape, they prefer to assemble in water into bilayer-based structures, such as vesicles, and lamellar © XXXX American Chemical Society

liquid crystals with near-zero curvature. The study on vesicles has increasingly become important because of their applications in drug and gene delivery.14−17 Kaler et al., in 1989, reported the spontaneous formation of thermodynamically stable vesicles in dilute aqueous solution of single-tailed cationic and anionic surfactant mixtures,18 which demonstrated a new path in the following years. Recently, a great deal of efforts has been paid to visualize the interaction and stability of vesicles in surfactant mixtures.19−21 However, to optimize the stable vesicle formation by the mixed systems, it is important to have a general understanding of the interplay of interactions between the surfactant in a mixed system and the factors that influence the vesicle stability. Although N-acylamino acidate (NAA) surfactants are used as detergents, their phase behavior in mixtures with cationic surfactants has not received much attention. In the present contribution, aggregation properties of aqueous solutions containing mixtures of the anionic sodium N-alkanoyl-L-alaninate (SAA) surfactants (see Figure 1 for structure) with cationic surfactant N-cetylpyridinium chloride (CPC) have been discussed. However, it is of interest to study the vesicle formation by such mixtures where anionic surfactant chain length is different. Also mixing composition of the two surfactants has a dramatic influence on the systems due to their effect on the electrostatic balance at the aggregate/solution Received: September 18, 2014 Revised: February 6, 2015 Accepted: February 6, 2015

A

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°C) unless otherwise mentioned. Temperature-controlled measurements were carried out by use of a Thermo Neslab RTE −7 circulating bath. 2.4. Solution Preparation. Samples were prepared by mixing the cationic and anionic surfactants at a desired concentration and different mixing ratio from the individual surfactant stock solution. After sealing, the samples were mixed by hand shaking, and the solutions were left to equilibrate at room temperature for few hours. The composition of the mixtures thus obtained is expressed in terms of molar fraction, X1 (= [SAA]/([SAA] + [CPC]) of SAA. The pH of the samples was varied using 20 mM phosphate buffer (having equal ionic strength of 0.0178 M). Since DPH is insoluble in water, a 1.0 mM stock solution of the probe in 20% (v/v) methanol−water mixture was prepared. The final concentration of the probe was adjusted to 1 μM by addition of an appropriate amount of the stock solution. Prior to observation and measurements, the solutions were allowed to keep at room temperature and were left standing for several hours to stabilize. 2.5. Turbidity Measurements. Stability of the vesicle was monitored by the turbidity measurements of the mixed surfactant system as a function of time.34 All turbidity (τ = 100 − %T) measurements were carried out at room temperature and recorded on a Shimadzu (model 1601) spectrophotometer at 450 nm using a quartz cell with a path length of 1 cm. The instrument was set at the transmittance (turbidity) mode. 2.6. Steady-State Fluorescence Measurements. Steadystate fluorescence anisotropy (r) of DPH probe (∼1 × 10−6 M) was measured on a PerkinElmer LS-55 luminescence spectrometer equipped with filter polarizers that uses the Lformat configuration.35 A quartz cell of 1 cm path length was used for all fluorescence measurements. The samples were excited at 350 nm, and fluorescence intensity was measured at 450 nm. A 430 nm cutoff filter was placed in the emission beam to eliminate the effects of scattered light, if any. The excitation and emission slit with bandpass were 2.5 and 7.5 nm, respectively. The software supplied by the manufacturer automatically determined the correction factor and anisotropy value. In all the cases, the anisotropy values were averaged over an integration time of 10 s and maximum number of five measurements for each sample. The temperature (30 ± 0.1 °C) of the water-jacketed cell holder was controlled by use of a Julabo F12 circulating bath. Since DPH is insoluble in water, a 0.5 mM stock solution of the probe in methanol−water mixture was prepared. The final concentration of the probe was adjusted to 1 × 10−6 M by addition of an appropriate amount of the stock solution. All the fluorescence measurements were started at least 2−3 h after the sample was prepared. 2.7. Dynamic Light Scattering. Sample solutions for DLS measurements were prepared by mixing the stock solutions of cationic and anionic surfactants in Milli Q water. DLS measurements were carried out by Zetasizer Nano ZS (Malvem Instrument Lab, Malvern, U.K.). The intensity autocorrelation functions were analyzed by cumulant36 method using the software provided by the manufacturer. Cylindrical glass cells were used. The scattering intensity was normally measured at θ = 173° to the incident beam but with some measurements at other angles to check the angular dependence of the decay rate (Γ) of the autocorrelation function. All DLS measurements were performed at room temperature, ∼25 °C. The average diffusion coefficient and corresponding hydrodynamic diameter of the particles were calculated using Stokes−Einstein37

Figure 1. Chemical structure of sodium N-alkanoyl-L-alaninate.

interface as well as the packing of the hydrophobic tails inside the vesicles. This mixing ratio provides an additional way of adjusting system properties in addition to total surfactant concentration, aging,22,23 pH,24,25 temperature,23−27 and cosolvents.28,29 Herein, the self-assembly in mixtures of four sodium N-(nalkanoyl) alaninates SOA (C8Ala), SDA (C10Ala), SLA (C12Ala), and STA (C14Ala) surfactants with different chain lengths and CPC were studied by transmission electron microscopy (TEM), confocal fluorescense microscopy (CFM), dynamic light scattering (DLS), and fluorescence spectroscopy techniques in different environments. Fluorescence probe technique has proved to be a powerful tool in the study of aggregation of pure and mixed surfactant systems30−32 because of its capacity for obtaining information regarding microenvironment of the aggregates. Considering the rich aggregation behaviors and the challenging characterization in both cationic- and anionic-rich regions of the corresponding systems, they have been compared and studied systematically.

2. EXPERIMENTAL SECTION 2.1. Materials. L-alanine and CPC were obtained from SRL, Mumbai, and fluorescence probes like 1,6-diphenyl-1,3,5hexatriene (DPH), 5(6)-carboxyfluorescein (CF) (Aldrich), and calcein were recrystallized in acetone−ethanol mixture at least three times and stored in a vacuum desiccator before use. Purity of all the probes was confirmed by the measurement of fluorescence emission as well as excitation spectra. All the reagents and solvents like ethanol, methanol, and acetone were of good quality commercially available and were purified and distilled fresh whenever required. Analytical grade sodium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium hydroxide were purchased from SRL, Mumbai. High-quality Milli Q (18.2 MΩ) water with pH of 7.4 (20 mM PBS) was used for solution preparation. 2.2. Synthesis. Sodium salts of SAA of different chain lengths were synthesized from n-acyl chloride and L-alanine according to the reported literature.33 The sodium salt was purified twice from ethanol−dry acetone mixture. The molecular structures of the compounds were determined by elemental analysis, 1H NMR, and IR spectra (see Supporting Information S1). 2.3. General Instrumentation. 1H NMR spectra were recorded on a Bruker SEM 200 instrument using TMS (trimethyl silane) as standard. Thermo Orion model 710A+ digital pH meter (EC India Ltd., Kolkata) using a glass electrode calibrated with a pH = 7.00 and pH = 4.01 buffer was used to measure the pH of the solutions. Conductivity measurements were carried out with a Thermo Orion 150A+ conductivity meter (calibrated with a KCl solution) that uses a cell having cell constant equal to 0.4665 cm−1. All the measurements were carried out at room temperature (∼30 B

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free dye. The apparent leakage of encapsulated dye from the aqueous core of the vesicle was determined by monitoring fluorescence intensity with time using PerkinElmer LS-55 luminescence spectrometer. The % leakage of calcein is thus defined as

equation for spherical particles. The polydispersity values (less than 0.3) for the particle reflect the intensity-weighted relative variance of the diffusion coefficient and should be close to zero for monodispersed systems. 2.8. Zeta Potential Measurements. The surface zeta potentials of the vesicles were also measured using Zetasizer Nano ZS (Malvern Instrument Laboratory, Malvern, U.K.) optical system equipped with an He−Ne laser operated at 4 mW (λo ≈ 632.8 nm) at 25 °C. The concentrations of the surfactants were different at two different ratios (X1 = 0.20 and 0.80) with pH of 7.4 (20 mM PBS). Three successive measurements were taken for each sample. 2.9. Transmission Electron Microscopy. The morphological structures of the aggregates were investigated by transmission electron microscope by negative straining method. A carbon coated copper grid was dipped in a drop of the aqueous surfactant solution for 1 min; excess solution was blotted with filter paper, air-dried for an hour, and then negatively stained with freshly prepared 1% (w/v) aqueous uranyl acetate. The specimens were kept in desiccators until use and were visualized on a JEOL-JEM 2100, Japan, transmission electron microscope operated at 200 kV at 25 °C. 2.10. Confocal Fluorescence Microscopy. CFM was employed to visualize dimensions of the dye-trapped vesicles. All CFM imaging experiments were performed in FV 1000 Olympus Confocal Microscopy instrument equipped with a laser scanning module (LSM) microscope and a PLAPON 60 X oil immersion objectives with numerical aperture (NA) of 1.42. For CF dye, the excitation light from lasers at 488 nm, which is reflected by a dichroic mirror (DM 4055/488), and the emission is split by another dichroic mirror (DM 405/488). For CF labeled vesicles, we used a 488 nm laser and a 520 nm filter. Microscope glass slides (Riviera, 25.4 mm × 76.2 mm) were treated with a dye trapped vesicle solution prior to use in order to prevent the vesicles from adhering to the glass coverslip. Dye was trapped into the vesicle by gentle mixing of surfactant and dye into methanol followed by rotary evaporation and making aqueous solution. The excess dye was removed by dialysis method using an ultrafiltration cellulose acetate membrane for biodialyser (pore size 10 kDa MWCO, Diam 16 mm) for 10− 12 h. An aliquot of the undiluted vesicle solution was pipetted into the glass slide and sealed with a coverslip and left to sit for a few minutes before analysis. All vesicles were imaged at room temperature, and image projections of both cationic- and anionic-rich dye-trapped vesicles were analyzed using FV10ASW 1.6 Viewer software. 2.11. Dye Entrapment and Vesicle Stability Determination. For entrapment of fluorescent dyes, such as calcein or CF within the vesicle, the required amount of surfactant, cholesterol, and dye in methanol solution were mixed properly, and then solvent was completely evaporated with a stream of N2 gas. The dry mass was soaked with a small amount of buffer overnight and then diluted with an appropriate volume of buffer (pH 7.4). The dye-entrapped vesicles thus obtained were separated from the dye molecules that were not encapsulated and were free in solution by size exclusion chromatography (SEC). A 2 cm × 22 cm column packed with Sephadex G50 resin (medium mesh) was used. In a typical experiment, a 200 μL (containing 30 mM surfactant mixture, 2 mM calcein, 10 mol % cholesterol, and 150 mM NaCl at pH 7.4) sample of vesicle solution was eluted through the column. The vesicle mixture was observed to divide into two clear bands, one containing the dye-bearing vesicles and the other consisting of

% leakage = 100 × (F − F0)/(Ft − F0)

(1)

where F is fluorescence intensity at given time, Ft is the total fluorescence measured after the disruption of vesicles with the addition of 2% Triton X, and F0 is the zero time fluorescence intensity at 511 nm. To avoid artifacts in fluorescence spectroscopy from light scattering or from dye aggregation inside the vesicles, the intensity of the encapsulated dye was determined after first disrupting the vesicle membranes by the addition of 2% Triton X-100 surfactant.

3. RESULTS AND DISCUSSION 3.1. Composition and Concentration-Dependent Aggregation Behavior. Changes in the molecular structure of the surfactants in the mixed surfactant system alter their physicochemical properties, which then results in changes in their applications. For anionic surfactants, the effects of increasing the length of the hydrophobic tail, while keeping headgroup constant, have an enormous effect on the interaction as well as on the self-assembly of mixed surfactant systems. However, to optimize the self-assembly behavior of the mixed system, solution were prepared at a concentration a few times greater than the mixed cmc. The individual cmc values of SOA, SDA, SLA, STA, and CPC are 81.5 mM, 46.0 mM, 11.7 mM, 3.05 mM, and 0.9 mM, respectively.6 Significantly, the mixed system cmc values of current system and similar type of catanionic mixtures are much lower compared to the pure ones at different compositions.21 To understand the aggregation behavior of the mixed system, fluorescence anisotropy was measured, which measures the anisotropy of DPH (aligned parallel to the lipid tails) due to changes in the degree of lipid ordering. Lipid ordering is related to the microviscosity or rigidity in the microenvironment of the probe,38,39 which is higher in the vesicular phase than the micellar phase. To confirm the formation of bilayer, fluorescence anisotropy of DPH was measured in the presence of mixed surfactants systematically. The steady-state fluorescence anisotropy (r) of DPH was measured in the presence of mixed surfactant systems in order to investigate both composition and concentration-dependent self-assembly of the SOA−CPC, SDA−CPC, SLA−CPC, and STA−CPC mixed systems. The composition-dependent variation of fluorescence anisotropy of DPH probe in cationic-, (X1 = 0.2) and anionic-rich (X1 = 0.8) mixtures of the binary systems has been shown in Figure 2. The representative plots showing variation of r-value with total surfactant concentration can be found in Figure 3. As in the case of the SLA−CPC system, the r-value is observed to be highest around X1 = 0.5. Similar value of the fluorescence anisotropy has been reported for liposomes.40,41 The decrease of r-value with the increase or decrease of molar fraction of anionic surfactant from 0.5 can be associated with vesicle-to-micelle transition (Figure 2). At a given composition, the large value of r in the case of STA−CPC compared to SOA−CPC system is consistent with higher rigidity of the bilayer membrane due to the hydrophobic chain length effect of anionic surfactant. The plots in Figure 3 also show a large initial increase of r-value with increasing total surfactant concentration above cmc values reaching a plateau at C

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accompanying deformation of membrane had until now been observed only in systems to which specific ligands and receptors were added. In such cases, no deformation occurs even when vesicles are brought together by a specific pHsensitive dye, such as CF. This kind of self-assembly of vesicles occurs when such dye molecules are entrapped into the aqueous core. The existence of aqueous core within the aggregates formed by the SOA−CPC, SDA−CPC, SLA−CPC, and STA−CPC systems was also confirmed primarily by the results of conductivity studies (see Supporting Information S2). 3.3. Hydrodynamic Diameters of Vesicles. The DLS measurements were performed to obtain mean hydrodynamic size of the vesicles formed by the mixed systems. The measured intensity distributions and hence hydrodynamic diameter (DH) of the aggregates of the mixed SDA−CPC systems for cationicand anionic-rich compositions have been presented in Figure 6, although the other mixed systems follow the same characteristics. The large sizes of the self-assemblies thus indicate formation of vesicles and are consistent with the results of TEM and CFM studies described above in this section. The mean DH values of the vesicular aggregates formed by different mixed systems can also be found in Table 1. The data in Table 1 compare various self-assembly parameters, such as cac, r-value, mean DH, zeta potential (ζ), etc., of the CnAla−CPC mixed systems with the anionic surfactant of different chain length. It is interesting to observe that the average DH values in both cationic-rich and anionic-rich compositions of the mixtures, within the experimental error limit, are equal in the case of SLA−CPC and STA−CPC systems, but for the SOA−CPC and SDA−CPC systems, the vesicle sizes are larger in the anionic-rich mixture compared to those in cationic-rich compositions. 3.4. Zeta Potential Measurements. The zeta potential (ζ) measurements were performed at different compositions of the mixed systems to determine the overall surface charge of the vesicles in the mixtures. However, the individual zeta potentials of SOA, SDA, SLA, STA, and CPC are −58.9(±1.3) mV, −64.3(±0.09) mV, −74.5(±0.07) mV, −81.2(±1.8) mV, and +68.6(±5.8) mV, respectively. It is observed that the surface charge (negative) of the vesicles in anionic-rich mixtures increases with the increase of chain length of the anionic surfactant. Similar behavior can also be observed with the cationic-rich (positive surface charge) mixtures, as shown in

Figure 2. Variation of fluorescence anisotropy (r) of DPH as a function of X1 of anionic surfactant in 1.0 mM SOA−CPC, SDA− CPC, SLA−CPC, and STA−CPC systems at 30 °C.

about 3−15 mM depending upon chain length of the CnAla surfactant. The concentration corresponding to the maximum r-value is lowest in the case of STA. As in the case of SLA− CPC system, increase of surfactant concentration above 30 mM resulted in a decrease of r-value (not shown in the figure) consistent with the transformation of vesicles to rod-like micelles. 3.2. Microscopic Studies. The TEM micrographs of negatively strained specimens prepared from aqueous CnAla− CPC surfactant solutions (X1 = 0.2) have been depicted in Figure 4. The micrographs reveal existence of large spherical vesicles that were spontaneously formed even in dilute aqueous solution. The internal diameters of the spherical vesicles are in the range of 50−160 nm (Figure 4D) in the case of SOA−CPC system in comparison to 40−100 nm for SLA−CPC system. In 1 mM SDA−CPC mixture (X1 = 0.2), coexistence of small as well as large vesicles can also be observed. The hydrodynamic size of the vesicles was further measured by DLS method as discussed later. The CFM images in Figure 5, panels A−D further confirm the morphology of the vesicles obtained by TEM and fluorescence techniques. The results clearly indicate that the mixed catanionic surfactant vesicles formed by CnAla−CPC system are spherical and hollow. The interesting phenomenon is that the vesicles are not deformed in a way that is usually observed in aggregates. The formation of vesicles without

Figure 3. Plot of fluorescence anisotropy (r) of DPH versus total [surfactant] of SOA−CPC, SDA−CPC, SLA−CPC, and STA−CPC systems at 30 °C. (A) X1 = 0.2, and (B) X1 = 0.8. D

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Figure 4. Negatively strained (1% uranyl acetate) transmission electron micrograph of (A) STA−CPC (1 mM, X1 = 0.2), (B) SLA−CPC (1 mM, X1 = 0.2), (C) SDA−CPC (1 mM, X1 = 0.2), and (D) SOA−CPC (2 mM, X1 = 0.2) mixtures.

Figure 6. Distributions of hydrodynamic diameter (DH) of vesicles in 1 mM SDA−CPC mixtures of different compositions.

3.5. Stability of Vesicles. 3.5.1. Effect of Aging. The stability of the spontaneously formed vesicles in aqueous solution of the surfactants was examined and followed spectrophotometrically by measuring the time dependence of turbidity (τ) at 450 nm. The results (see Supporting Information S3) indicated a slight increase of turbidity with time. However, the scattering was observed to be most pronounced in the case of 20 mM STA−CPC system and least in the case of SOA−CPC (20 mM) system, which is consistent with the vesicle size in these mixtures. This means that the vesicles formed from STA−CPC systems are larger

Figure 5. Confocal fluorescence microscopic images of CF-trapped vesicle formed by 20 mM (A) SOA−CPC, (B) SDA−CPC, (C) SLA− CPC, and (D) STA−CPC catanionic mixtures with X1 = 0.2.

Table 1. It is worthy to mention that high absolute value of ζ potential in two different compositions (X1 = 0.2 and 0.8) indicates that strong interactions exist among the mixedsurfactant vesicles, and hence the stability of the vesicles can be manifested. E

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Table 1. Critical Aggregation Concentration (cac), Fluorescence Anisotropy (r), Mean Hydrodynamic Diameter (DH), and Zeta Potential (ζ) for SOA−CPC (2 mM, X1 = 0.2 and 0.8) and SDA−CPC, SLA−CPC, STA−CPC (1 mM, X1 = 0.2 and 0.8) in Aqueous Phosphate Buffer Solution (pH 7.4) SOA−CPC

SDA−CPC

SLA−CPC

STA−CPC

physical properties

X1 = 0.2

X1 = 0.8

X1 = 0.2

X1 = 0.8

X1 = 0.2

X1 = 0.8

X1 = 0.2

X1 = 0.8

cac (mM) anisotropy (r) DH (nm) ζ (mV)

0.38 0.177 128 ± 7 +21.3 ±1.5

0.51 0.126 144 ± 7 −19.8 ±2

0.149 0.164 158 ± 5 +32.2 ±3

0.168 0.165 172 ± 6 −21.6 ±1.5

0.091 0.172 250 ± 8 +38.8 ±2

0.085 0.195 232 ± 5 −29.2 ±2.5

0.043 0.221 246 ± 3 +24.5 ±1.5

0.049 0.216 236 ± 2 −37.2 ±3.5

surfactant mixtures as seen in Figure 8, panel A. The large increase in r-value upon increase of [Chol] suggests increase of rigidity of vesicle membrane. This is due to the fact that cholesterol lowers membrane permeability and imparts better stability or rigidity of the vesicular aggregates. 3.5.3. Effect of pH. The pH dependence of r-value of DPH probe in the presence of surfactant mixtures of CnAla−CPC systems has been shown by the corresponding plots in Figure 8, panel B. The r-value is found to decrease with the decrease in pH of the solution. All the plots exhibit a sigmoid curve corresponding to two-state process. The changes of anisotropy are almost similar for all the systems and can be attributed to protonation of the −COO− group that reduces ionic interaction and thereby facilitates vesicle-to-micelle transition (see Supporting Information S4). The pKa values of the individual surfactants are slightly lower than those of the corresponding mixture of the surfactant system in aqueous solution. Similar increase in pKa values upon aggregation has been also suggested for fatty acids by other researchers.42−45 Thus, it can be concluded that vesicle structures are more favored at pH above their respective pKa value. 3.5.4. Effect of Temperature. The temperature effect on the vesicle stability was followed by measurement of fluorescence anisotropy of DPH. The plot of the variation of r as a function of temperature is shown in Figure 9, panel A. The r-value is high at low temperature, but it decreases with the rise in temperature due to phase transition between the gel-like states to the liquid−crystalline state. The phase transition temperatures, Tm, are greater than 37 °C, which suggests higher stability of the vesicles. The phase transition temperatures were also measured in the presence of Chol, which has been shown to increase rigidity of vesicle bilayer. Thus, as seen in Figure 9, panel B, the Tm increases upon addition of 10% (w/v) Chol in

compared to those by SLA−CPC, SDA−CPC, and SOA−CPC systems for a fixed composition (see Table 1). To substantiate the results of turbidity measurements, the time dependence of the hydrodynamic size of the vesicles in solution was further followed by DLS method. Figure 7 shows the size distributions

Figure 7. Histograms of size distributions in 1 mM SDA−CPC mixtures at different time intervals. (A) X1 = 0.2, and (B) X1 = 0.8.

of the vesicles in both cationic-rich and anionic-rich mixtures of SDA−CPC system measured at different time intervals. As observed, the size of the vesicles changes only slightly upon standing up to 60 days, which is attributed to the fact that stable vesicles are formed. 3.5.2. Effect of Cholesterol. Cholesterol also potentially modulates the hydrocarbon chain fluidity of the lipid membrane. The membrane stabilizing effect of cholesterol (Chol) is demonstrated by the increase fluorescence anisotropy value of DPH probe with increasing Chol concentration in the

Figure 8. Variation of fluorescence anisotropy of DPH with (A) [Chol] in 1 mM (X1 = 0.2 and 0.8) SLA−CPC and STA−CPC mixtures and (B) pH in different surfactant mixtures (X1 = 0.2) at 30 °C. F

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Figure 9. Temperature dependence of fluorescence anisotropy of DPH in SOA−CPC, SDA−CPC, SLA−CPC, and STA−CPC mixtures (1 mM, X1 = 0.2) (A) in absence of Chol and (B) in the presence of 10 mol % Chol.

Figure 10. (A) Fluorescence spectra of vesicle-entrapped calcein in 20 mM SLA−CPC (X1 = 0.2) containing 10 mol % Chol at different pH. (B) Plot of extent of calcein released from SLA−CPC (X1 = 0.2) mixed vesicles at pH 7.4 as a function of time.

in cationic-rich (X1 = 0.2) mixture of SLA−CPC system in buffered solution of desired pH. Because of the decrease in solution pH, vesicles disrupt and calcein is released as shown by the quenching of fluorescence intensity (Figure 10A). The entrapment and slow release of water-soluble drug is thus demonstrated by the vesicles in SLA−CPC mixture. The long-term stability of the dye-encapsulated vesicles was also considered. As time progresses, it is expected that the encapsulated dye would leak through the vesicle bilayer into the bulk solution. Because of self-quenching, the fluorescence intensity of vesicle-entrapped dyes is very weak. However, as discussed earlier, the release of calcein from the vesicle core will enhance fluorescence intensity.6 The time-dependent increase of the fluorescence emission intensity of calcein was taken as a direct measure of the calcein efflux rates from vesicles. This is illustrated in Figure 10, panel B, which shows the time trace obtained over the course of two months. Finally, the disruption was observed to be completed with the addition of Triton X100, a nonionic detergent that disrupts both lipid and surfactant vesicles.13 Figure 10, panel B shows the % leakage of entrapped calcein from the vesicles formed by the cationic-rich SLA−CPC mixture (X1 = 0.2). It is seen that only 40% of the entrapped dye molecules were released during the period of observation. The relatively slow release suggests that dye entrapment and its subsequent release are not influenced largely with time. 3.7. Effect of Chain Length of the Anionic Surfactant on Vesicle Formation. From the above experiments, a good correlation can be observed between fluorescence anisotropy data and chain length of the anionic surfactant in different CnAla−CPC systems. To further compare the stability of the spontaneously formed vesicles in CnAla−CPC mixed systems, anisotropy of DPH probe in solutions containing different

mixtures of SOA−CPC, SDA−CPC, SLA−CPC, and STA− CPC systems. Temperature-dependence anisotropy in the presence of 10% (w/v) Chol follows the same trend as in the absence of additives into it. Although the natures of r versus T dependences are generally similar, the order parameters differ significantly from one system to the other. The phase transition temperatures (Tm) for different SAA−CPC systems also change when cholesterol is doped into it. From the plot in Figure 9, panel A, it is observed that for SOA−CPC system, Tm is lower compared to other systems, and it follows in this order: SOA− CPC < SDA−CPC < SLA−CPC < STA−CPC system, although in these systems, Tm is greater than 37 °C. When 10% (w/v) cholesterol is added in the vesicular system containing 1 mM (X1 = 0.2) SAA−CPC mixture, anisotropy shows a higher value also in higher temperatures. Tm is also increased, and consequently a broadening effect of the phase transition process occurs. All the profiles in these systems indicated inflections pertaining to their thermotropic phase transition processes and their temperature-dependence anisotropy, which are described in our earlier investigation.13 3.6. Vesicle Stability and pH-Induced Release of Model Drugs. To investigate drug entrapment ability and pH-induced release by the vesicle structures, calcein was used as a model drug. The pH-sensitive dye calcein was chosen as its four carboxylic groups get deprotonated at high pH. The tetra anion due to its low permeability remains entrapped inside the vesicles during these measurements. Consequently, fluorescence intensity of calcein decreases with the decrease of solution pH and also with time. Fluorescence spectra (Figure 10A) of entrapped calcein (λex = 495 nm and λem = 511 nm) were measured immediately after dilution of the vesicle phase G

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Central Research Facility, IIT Kharagpur, India; Dr. S. K. Ghosh, Department of Biotechnology, IIT Kharagpur, India; and Dr. J. Dey, Department of Chemistry, IIT Kharagpur, India for instrumental assistance.

surfactant mixtures was measured at two different compositions (X1 = 0.2 and 0.8) at a constant concentration of 5 mM. The results have been included in Table 1 (at a fixed concentration of 5 mM). The variation of chain length (Cn) of the anionic surfactant has a large effect on the stability as well as size of the vesicles in catanionic mixtures (see Supporting Information S5). As discussed for this system earlier, the vesicles formed by STA−CPC system have greater membrane rigidity and hence enhanced stability. Thus, on the basis of the r-values, it can be concluded that the stability of vesicles formed by CnAla−CPC systems decreases in the order of STA−CPC > SLA−CPC > SDA−CPC > SOA−CPC. It should also be noted that for these systems, irrespective of the chain length of the CnAla surfactant, the vesicles in cationic-rich mixture are more stable than in anionic-rich mixtures. Significantly, it is observed that cationicrich vesicles are more stable for systems with Cn ≤ 10, whereas anionic-rich vesicles are more stable for systems with Cn ≥ 12.

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ASSOCIATED CONTENT

S Supporting Information *

Conductivity studies, turbidity studies, and DLS size distribution. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Rodríguez-Pulido, A.; Casado, A.; Muñoz-Ubeda, M.; Junquera, E.; Aicart, E. Experimental and theoretical approach to the sodium decanoate−dodecanoate mixed surfactant system in aqueous solution. Langmuir 2010, 26, 9378−9385. (2) Bera, A.; Ojha, K.; Mandal, A. Synergistic effect of mixed surfactant systems on foam behavior and surface tension. J. Surfactants Deterg. 2013, 16, 621−630. (3) Cid, A.; Morales, J.; Mejuto, J. C.; Briz-Cid, N.; Rial-Otero, R.; Simal-Gandara, J. Thermodynamics of sodium dodecyl sulphate− salicylic acid based micellar systems and their potential use in fruits postharvest. Food Chem. 2014, 151, 358−363. (4) Kabir-ud-Din; Rub, M. A.; Naqvi, A. Z. Mixed micelles of amphiphilic drug promethazine hydrochloride and surfactants (conventional and gemini) at 293.15 K to 308.15 K: Composition, interaction, and stability of the aggregates. J. Colloid Interface Sci. 2011, 354, 700−708. (5) Ota, S.; Yoshizawa, S.; Takeuchi, S. Microfluidic formation of monodisperse, cell-sized, and unilamellar vesicles. Angew. Chem., Int. Ed. 2009, 48, 6533−6577. (6) Patel, A. R.; Joshi, V. Y. Evaluation of SLS: APG mixed surfactant systems as carrier for solid dispersion. AAPS Pharm. Sci. Tech. 2008, 9, 583−590. (7) Kalyansundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press Inc.: Orlando, FL, 1986. (8) Zana, R. In Surfactant Solutions: New Methods of Investigation; Marcel Dekker: New York, 1987; p 241. (9) Miyagishi, S.; Suzuki, H.; Asakawa, T. Microviscosity and aggregation number of potassium N-acylalaninate micelles in potassium chloride solution. Langmuir 1996, 12, 2900−2905. (10) Zhao, L.; Liu, J.; Zhang, L.; Gao, Y.; Zhang, Z.; Luan, Y. Selfassembly properties, aggregation behavior, and prospective application for sustained drug delivery of a drug-participating catanionic system. Int. J. Pharm. 2013, 452, 108−115. (11) Zhang, L.; Liu, J.; Tian, T.; Ji, X.; Li, Z.; Luan, Y. Pharmaceutically active ionic liquid self-assembled vesicles for application as an efficient drug delivery system. ChemPhysChem 2013, 14, 3454−3457. (12) Ghosh, S.; Ambade, B.; Ray, A. Stable catanionic vesicles as drug delivery vehicle. Sci. Adv. Mater. 2013, 5, 1837−1846. (13) Ghosh, S.; Dey, J. Interaction of sodium N-Lauroylsarcosinate with N-alkylpyridinium chloride surfactants: Spontaneous formation of pH-responsive, stable vesicles in aqueous mixtures. J. Colloid Interface Sci. 2011, 358, 208−216. (14) Soussan, E.; Cassel, S.; Blanzat, M.; Rico-Lattes, I. Drug delivery by soft matter: Matrix and vesicular carriers. Angew. Chem. Int. Ed. 2009, 48, 274−288. (15) Consola, S.; Blanzat, M.; Perez, E.; Garrigues, J. C.; Bordat, P.; Rico-Lattes, I. Design of original bioactive formulations based on sugar−surfactant/non-steroidal anti-inflammatory catanionic self-assemblies: A new way of dermal drug delivery. Chem.Eur. J. 2007, 13, 3039−3047. (16) Verma, G.; Hassan, P. A. Self assembled materials: Design strategies and drug delivery perspectives. Phys. Chem. Chem. Phys. 2013, 15, 17016−17028. (17) Danoff, E. J.; Wang, X.; Tung, S. H.; Sinkov, N. A.; Kemme, A. M.; Raghavan, S. R.; English, D. S. Surfactant vesicles for highefficiency capture and separation of charged organic solutes. Langmuir 2007, 23, 8965−8971.

4. CONCLUSIONS The present study reveals the formation of stable catanionic vesicular aggregates of cationic CPC and anionic SAA surfactant mixtures in different compositions and concentrations. The observed results successfully demonstrate the formation of large spherical vesicular aggregates along with the presence of some smaller aggregates above the cmc of the surfactant mixture, which was confirmed by fluorescence anisotropy, TEM, DLS, and CFM studies as well. Fluorescence anisotropy studies reveal the vesicle formation with the increasing surfactant concentration and near equimolarity. The binary mixtures of amino acid−based surfactants with CPC have hydrodynamic diameter in the range of 20−250 nm, and the size of the particles remains almost the same even after two months of aging. Zeta potentials of the vesicles were found to be large in magnitude, and the observed long-term stability of the vesicles may be attributed to such high zeta potentials. The catanionic vesicles exhibited drug entrapment ability, and their stability at room temperature was found to be a longer period of time. However, with the decrease in pH (pH ≤ 5), the mixed surfactant vesicles are transformed into small mixed micelles. This means that the vesicles are sensitive to pH change of the environment. These pH-sensitive vesicles are potentially interesting as drug delivery system in which one aims to trigger the drug release from the vesicles as the pH is decreased from a neutral or slightly alkaline pH to an acidic pH. The lifetime of vesicles and micelles can therefore be controlled by varying the composition of the binary surfactant solutions and by an additive like cholesterol. Control of the amount of additive, temperature, solution pH, etc. phenomena is of importance for a large number of industrial processes where formulations must be tuned.

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DOI: 10.1021/ie503697c Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research (18) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasdzinski, J. A. N. Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants. Science 1989, 245, 1371−1374. (19) Nan, Y.; Hao, L. Mixed cationic and anionic surfactant systems. Prog. Chem. 2007, 19, 1291−1300. (20) Ghosh, S.; Ray, A. Alkyl chain length asymmetry effects of mixed N-acyl sarcosinate and N-cetylpyridinium chloride surfactants: Spontaneous formation of stable nanovesicles as excipient. Colloids Surf., A 2014, 461, 248−257. (21) Fernendes, R. M. F.; Marques, E. F.; Silva, B. F. B.; Wang, Y. Micellization behavior of a catanionic surfactant with high solubility mismatch: Composition, temperature, and salt effects. J. Mol. Liq. 2010, 157, 113−118. (22) Marques, E.; Khan, A.; Miguel, M. G.; da Lindman, B. Selfassembly in mixtures of a cationic and an anionic surfactant: The sodium dodecyl sulfate−didodecyldimethylammonium bromide− water system. J. Phys. Chem. 1993, 97, 4729−4736. (23) Kondo, Y.; Uchiyama, H.; Yoshino, N.; Nishiyama, K.; Abe, M. Spontaneous vesicle formation from aqueous solutions of didodecyldimethylammonium bromide and sodium dodecyl sulfate mixtures. Langmuir 1995, 11, 2380−2384. (24) Tsuchiya, K.; Sakai, H.; Saji, T.; Abe, M. Electrochemical reaction in an aqueous solution of a ferrocene-modified cationic surfactant mixed with an anionic surfactant. Langmuir 2003, 19, 9343− 9350. (25) Bergström, M.; Pedersen, J. S.; Schurtenberger, P.; Egelhaaf, S. U. Small-angle neutron scattering (SANS) study of vesicles and lamellar sheets formed from mixtures of an anionic and a cationic surfactant. J. Phys. Chem. B 1999, 103, 9888−9897. (26) Hao, J.; Yuan, Z.; Liu, W.; Hoffmann, H. In situ vesicle formation by a kinetic reaction in aqueous mixtures of single-tailed catanionic surfactants. J. Phys. Chem. B 2004, 108, 5105−5112. (27) Tondre, C.; Caillet, C. Properties of the amphiphilic films in mixed cationic−anionic vesicles: A comprehensive view from a literature analysis. Adv. Colloid Interface Sci. 2001, 93, 115−134. (28) Fendler, J. H. Membrene Mimetic Approach to Advanced Materials; Spriner-Verlag: Berlin, 1992. (29) Yu, W. L.; Pei, J.; Huang, W.; Zhao, G. X. Formation of CdS nanoparticles in mixed cationic−anionic surfactant vesicle system. Mater. Chem. Phys. 1997, 49, 87−92. (30) Hierrezuelo, J. M.; Aguiar, J.; Carnero Ruiz, C. Micellar properties of a mixed surfactant system constituted by n-octyl-β-dthioglucopyranoside and sodium dodecyl sulphate. Colloids Surf., A 2005, 264, 29−36. (31) Sehgal, P.; Kosaka, O.; Doe, H.; Otzen, D. E. Interaction and stability of mixed micelle and monolayer of nonionic and cationic surfactant mixtures. J. Dispersion Sci. Technol. 2009, 30, 1050−1058. (32) Shao, G.; Yang, Z.; Wang, M.; Zhao, Q. Proceedings of the 2nd International Conference on Asian-European Environmental Technology and Knowledge Transfer, Heifei, P.R. China, 2008; pp 420−423. (33) Mhaskar, S. Y.; Prasad, R. B. N.; Laksminarayana, G. Synthesis of N-acyl amino acids and correlation of structure with surfactant properties of their sodium salts. J. Am. Oil Chem. Soc. 1990, 67, 1015− 1019. (34) Bergstrom, M. Thermodynamics of vesicle formation from a mixture of anionic and cationic surfactants. Langmuir 1996, 12, 2454− 2463. (35) Kang, K.; Kim, H.; Lim, K. Phase behavior and spontaneous vesicle formation in aqueous solutions of anionic ammonium dodecyl sulfate and cationic octadecyl trimethyl ammonium chloride surfactants. Bull. Korean Chem. Soc. 2007, 28, 667−674. (36) Vlachy, N.; Merle, C.; Touraud, D.; Schmidt, J.; Talmon, Y.; Heilmann, J.; Kunz, W. Spontaneous formation of bilayers and vesicles in mixtures of single-chain alkyl carboxylates: Effect of pH and aging and cytotoxicity studies. Langmuir 2008, 24, 9983−9988. (37) Renoncourt, A.; Vlachy, N.; Bauduin, P.; Drechsler, M.; Touraud, D.; Verbavatz, J. M.; Dubois, M.; Kunz, W.; Ninham, B. W. Specific alkali cation effects in the transition from micelles to vesicles through salt addition. Langmuir 2007, 23, 2376−2381.

(38) Mata, J.; Varade, D.; Ghosh, G.; Bahadur, P. Effect of tetrabutylammonium bromide on the micelles of sodium dodecyl sulfate. Colloids Surf., A 2004, 245, 69−73. (39) Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy; Pecora, R., Ed.; Springer: New York, 1985. (40) Cogan, U.; Schinitzky, M.; Weber, G.; Nishida, T. Microviscosity and order in the hydrocarbon region of phospholipid and phospholipid−cholesterol dispersions determined with fluorescent probes. Biochemistry 1973, 12, 521−528. (41) Zachariasse, K. A.; Kuhnle, W.; Weller, A. Intramolecular excimer fluorescence as a probe of fluidity changes and phase transitions in phosphatidylcholine bilayers. Chem. Phys. Lett. 1980, 73, 6−11. (42) Walde, P.; Wick, R.; Fresta, M.; Mangone, A.; Luisi, P. L. Autopoietic self-reproduction of fatty-acid vesicles. J. Am. Chem. Soc. 1994, 116, 11649−11654. (43) Bachmann, P. A.; Luisi, P. L.; Lang, J. Autocatalytic selfreplicating micelles as models for prebiotic structures. Nature 1992, 357, 57−59. (44) Cistola, D. P.; Small, D. M. Micelle formation and phase separation. J. Am. Chem. Soc. 1990, 112, 3214−3215. (45) Cistola, D. P.; Hamilton, J. A.; Jackson, D.; Small, D. M. Ionization and phase behavior of fatty acids in water: Application of the Gibbs phase rule. Biochemistry 1988, 27, 1881−1888.

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DOI: 10.1021/ie503697c Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX