Sodium

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Langmuir 1998, 14, 590-594

Kinetics of Formation of Vesicles from Lecithin/Sodium Xylenesulfonate Micelles from Stopped-Flow Measurements Samuel E. Campbell, Zhiqiang Zhang, Stig E. Friberg,* and Ramesh Patel Department of Chemistry, Clarkson University, Potsdam, New York 13699-5810 Received July 11, 1997. In Final Form: November 5, 1997 The kinetics of vesicle formation from hydrotrope/lecithin micelles in the system of lecithin, sodium xylenesulfonate (SXS), and water were studied via the stopped-flow process in conjunction with light scattering. The essential phases in the system are a micellar (L1) phase and a lamellar liquid-crystalline (LLC) phase. Based upon light scattering and conductance measurements in the L1 phase, the ratio of SXS to lecithin molecules in the micelles was approximately 1. The results of the stopped-flow process showed the mechanism of formation for the vesicles, which lie in equilibrium between the L1 and LLC phases, to be by the agglomeration of fragments.

Introduction The study of associating amphiphilic systems, which form complex molecular structures including micelles, vesicles, liquid crystals, and microemulsion droplets, has been undertaken due to the wide-ranging applicability and versatility of these systems which have made them ubiquitous in modern life.1-6 The vesicles7 have attracted special attention because of their occurrence in medical8-10 and personal care applications,11,12 and their preparation,13-15 structure,16-18 and stability19 have been intensely studied.20 * Author to whom correspondence is addressed. Phone: 315268-6500. Fax: 315-268-7990. E-mail: [email protected] (1) Zana,R., Ed. Surfactant Solutions, New Methods of Investigation; Surfactant Science Series Vol. 22; Marcel Dekker: New York, 1987. (2) Scamehorn, J.F., Harwell, J.H., Eds. Surfactant-Based Separation Processes; Surfactant Science Series Vol. 33; Marcel Dekker: New York, 1989. (3) Bender, M., ed. Interfacial Phenomena in Biological Systems; Surfactant Science Series Vol. 39; Marcel Dekker: New York, 1991. (4) Friberg, S. E., Lindman, B., Eds. Organized Solutions, Surfactants in Science and Technology; Surfactant Science Series Vol. 44; Marcel Dekker: New York, 1992. (5) Sjo¨blom, J., ed. Emulsions and Emulsion Stability; Surfactant Science Series Vol. 61; Marcel Dekker: New York, 1996. (6) Solans, C., Kunieda, H., Eds. Industrial Applications of Microemulsions; Surfactant Science Series Vol. 66; Marcel Dekker: New York, 1997. (7) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, The Netherlands, 1993. (8) Lasic, D. D., Martin, F., Eds. Stealth Liposomes; CRC Press: Boca Raton, FL, 1995. (9) Gregoriadis, G.; Wang, Z.; Bareholz, Y.; Francis, M. J. Immunology 1993, 80, 535. (10) Wichert, B. V.; Gonzalez-Rothi, R. J.; Straub, L. E.; Wichert, B. M.; Schreier, H. Int. J. Pharm. 1992, 78, 227. (11) Simonnet, J. T. Cosmet. Toiletries 1994, 109, 45. (12) Gareiss, J.; Fussbroich, P.; Ghyxzy, M. SOFW J. 1994, 120, 93. (13) Kaneko, T.; Sagitani, H. Colloids Surf. 1992, 69, 125. (14) Kaler, E. W.; Kamalakara Murthy, A.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371. (15) Trevino, L.; Fre´zard, F.; Rolland, J. P.; Postel, M.; Riess, J. G. Colloids Surf. 1994, 88, 223. (16) Jones, M. N. Adv. Colloid Interface Sci. 1995, 54, 93. (17) Scho¨nfelder, E.; Hoffmann, H. Ber. Bunsen-Ges. Phys. Chem. 1994, 98, 842. (18) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299. (19) Rusanov, A. I. Colloids Surf., A, 1993, 76, 7. (20) Rosoff, M. Ed. Vesicles; Surfactant Science Series Vol. 62; Marcel Dekker: New York, 1996.

One of the widely studied and utilized vesicle-forming surfactants is that of lecithin which is a double-chained phosphatidylcholine occurring naturally in the cell membranes of both plants and animals.21 Studies of lecithin, which have been done for at least 200 years,22 include the biological function23 and the use of lecithin in drug delivery systems.24 Lecithin has found a wide range of applications exploiting the surface-active nature of the molecule in conjunction with the biocompatibility of the compound.25 A second class of compounds, which has been widely used but only recently studied in detail is that of the hydrotrope, which is a small “blocklike” molecule. It does not per se strongly interact with an interface but shows extremely high solubilization of a surfactant26,27 or a hydrophobic amphiphile. One of the fundamental properties in the study of surfactant systems is the kinetics of formation, which have been extensively studied for micelles, microemulsions, and liquid crystals28-30 but which for vesicles have received little attention until recently31,32 with the examination of a system of surfactant and hydrotrope.33 Using the unique phase behavior of the hydrotropic system in conjunction with the shear properties encountered in a stopped-flow (21) Hill, H. E. Introduction to Lecithin; Nash Publ., Los Angeles, 1972. (22) Fourcroy, A. F. Ann. Chim. (Paris) 1793, 16, 282. (23) Hanin, I., Ansell, G. B., Eds. Lecithin: technological, biological, and therapeutic aspects; Plenum Press: New York, 1987. (24) Peeters, H., Ed. Phosphatidylchole: biochemical and clinical aspects of essential phospholipids; Springer-Verlag: Berlin, 1976. (25) Szuhaj, B. F. Ed. Lecithins: sources, manufacture, and uses; American Oil Chemists’ Society: Champaign, IL, 1989. (26) Balusubramanian, D.; Friberg, S. E. In Surface Colloid Science; Matijevic´, E., Ed.; Plenum Press: New York, 1993; Vol. 15. (27) Friberg, S. E.; Brancewicz, C.; Morrison, D. Langmuir 1994, 10, 2945. (28) Aniansson, E. A. G.; Wall, S. N. J. Phys. Chem. 1974, 78, 1024. (29) Aniansson, E. A. G.; Wall, S. N.; Almgren, M.; Hoffmann, H.; Kielmann, I.; Ulbricht, W.; Zana, R.; Lang, J.; Tondre, C. J. Phys. Chem. 1976, 80, 905. (30) Zana, R. In Dynamic Properties of Interfaces and Association Structures; Pillai, V., Shah, D. O., Eds.; American Oil Chemists’ Society: Champaign, IL, 1996; p 142. (31) Campbell, S.; Yang, H.; Patel, R.; Friberg, S. E.; Aikens, P. A. Colloids Polym. Sci. 1997, 275, 303. (32) Friberg, S. E.; Campbell, S.; Fei, L.; Yang, H.; Aikens, P. A.; Patel, R. Colloids Surf. (in press). (33) Friberg, S.E.; Yang, H.; Fei, L.; Sadasivan, S.; Rasmusen, D. H.; Aikens, P. A. J. Dispersion Sci. Technol. (in press).

S0743-7463(97)00774-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/14/1998

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experiment, the kinetics of vesicular formation were clarified in a nonionic surfactant system.31,32 To some degree unexpectedly, the study showed the formation of vesicles to take place by addition of single molecules to vesicle fragments. The formation of vesicles is obviously an important problem, and we found an investigation into lecithin vesicles to be of general interest based on the importance of lecithin in pharmaceutical development.8-10 With this paper, we examine the kinetics of formation for vesicles in a system of egg lecithin in combination with a model hydrotrope (sodium xylenesulfonate, SXS). Experimental Section Chemicals. L-R-Phosphatidylcholine (egg lecithin grade purified for intravenous injection, Kabi, Sweden) was used as received after storage under nitrogen at -20 °C. Sodium xylenesulfonate (Aldrich Chemical Co., Milwaukee, WI, less than 9% Na2SO4) was rinsed thrice with hexane, twice with acetone, and twice with ethanol. Following filtration, SXS was dried under vacuum for 72 h. Water was deionized. Small-Angle X-ray Diffraction. A small amount of the sample was drawn into a glass capillary of 0.5 mm in diameter, sealed at both ends, and placed in a brass sample holder which had a hole diameter of 2 mm. The X-ray radiation is Cu KR filtered by nickel foil to yield a wavelength of 0.1542 nm at 40 kV and 18 mA. The system is a Siemens Crystalloflex 4 using a Kiessig low-angle camera (Richard Seifert) and an ORDELA detection system, which allows the angle 2θ to range between 0.7 and 5.7°. The temperature in the X-ray sample chamber is controlled to (1 °C. The instrument is calibrated using lead stearate with an interlayer spacing of 4.82 nm. Phase Determination. The phase boundaries are determined by optical observation of the sample both visually and with the aid of a microscope. The presence of liquid crystals is determined visually by examination of the sample placed between crossed polarizing films. The liquid-crystalline region was confirmed through the use of low-angle X-ray diffraction. Tie lines were determined by matching the index of refraction in the singlephase regions. Electrical Conductivity. Electrical conductance measurements were performed on an ATI Orion (Boston, MA) Model 170 conductivity meter. Measurements were performed under ambient lab conditions of 50% relative humidity and 22 °C. Stopped-Flow Apparatus. The apparatus in which the stoppedflow measurements were made has been extensively described elsewhere.34 The measurements were performed using 550 nm light passed through a 2 mm inlet and based upon 90° light scattering. Dynamic Light Scattering. Light scattering measurements were performed on a Brookhaven Instruments system. The light source is an argon ion laser (Model 85, Lexel Laser) operating at a wavelength of 514.5 nm. The sampling setup consists of a BI-DS photomultiplier tube, a stepper-motor-controlled goniometer (BI-2000SM) and a BI-2030AT digital correlator. The dynamic light scattering (DLS) was performed at 90° with a sample maintained at a constant 23.0 ( 0.5 °C temperature.

Results and Discussion The essential parts of the phase diagram for the sodium xylenesulfonate, egg lecithin, and water system are presented in Figure 1. The system has a region of micelles (L1) and a lamellar liquid-crystalline regime (LLC). The phase behavior of this system is typical of that observed for a system composed of a surfactant (lecithin) and a hydrotrope (sodium xylenesulfonate).33 The feature of note in the basic phase diagram is the relatively high solubility (up to 22 wt % lecithin) achieved by the addition of the SXS hydrotrope. The ratio of solubilizer hydrotrope (SXS) to that of the vesicle-forming surfactant (lecithin) is (34) Hsu, W.; Patel, R.; Matijevic´, E. Appl. Spectrosc. 1987, 41, 402.

Figure 1. Phase diagram for the system water, egg lecithin, and sodium xylenesulfonate. L1 ) isotropic solution of SXS/ lecithin combination micelles. LLC ) lamellar liquid crystal. Inset: Composition of initial solutions (1-5) and final vesicle solutions (A-E) within the two-phase region L1-LLC. The vesicles were formed by combining an equal amount of water and a solution with L1. Table 1. Light Scattering Results on the Micellar Boundarya lecithin (wt %)

SXS (wt %)

water (wt %)

diameter 1 (nm)

diameter 2 (nm)

1.0 3.0 8.0 13.0 17.0

20.7 20.8 21.1 21.1 22.1

78.3 76.2 70.9 65.9 60.9

23.6 ( 2.4 15.6 ( 1.9 13.1 ( 2.0 9.5 ( 1.8 7.5 ( 1.8

24.0 ( 2.5 20.2 ( 2.4 23.2 ( 3.1 20.5 ( 3.0 23.3 ( 3.2

a Diameter 1 is measured assuming no bound SXS, while Diameter 2 is measured with the amount of SXS bound based upon conductivity measurements. The interpretation of the light scattering data was undertaken using the CONTIN method.

Table 2. Stopped-Flow Measurement Resultsa sample

lecithin (wt%)

size (nm)

τ (sec)

stability

A B C D E

1.5 2.5 4.0 5.5 6.5

57 ( 8 102 ( 21 319 ( 58 459 ( 71 502 ( 74

9.3 ( 3.1 4.9 ( 1.8 4.3 ( 2.1 3.1 ( 1.4 1.1 ( 0.6

>3 mo 9 weeks 2 weeks 3 days 5h

a The final concentration of sodium xylenesulfonate in all samples is 11 wt %. The decay time reported is based upon the average of an exponential fit to at least 15 individual runs. The stability is defined as when appreciable phase separation is observed.

approximately 1:1 in this case. This is a significant degree of solubilization for the phospholipid lecithin though even greater degrees of solubilization have been reported when combining a hydrotrope with a nonionic surfactant.33 In addition, the fact should be noted that the increase in solubilization takes place in the L1 region, which is in equilibrium with the lamellar liquid-crystalline phase (LLC) as indicated by the tie lines in Figure 1. The inset in Figure 1 indicates the initial and final concentrations that were studied using the stopped-flow method. The initial points (indicated by numerals) are micellar in structure, while the final points (indicated by characters) are in the two-phase region between the L1 micelle phase and the LLC phase, which in the stoppedflow process initially produces vesicular structures, which phase separate at time scales depending upon concentration as reported later and in Table 2. It may be mentioned that the thermodyanamically stable state in this part of the system is in the form of two phases: a lamellar liquid crystal in equilibrium with an aqueous solution of sodium

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Figure 2. Conductivity measured in the egg lecithin, SXS, and water system in the L1 region: (9) 31 wt % SXS; (0) 34 wt % SXS; (O) 43 wt % SXS. The lines are guides to the eye.

xylenesulfonate. Slow mixing of the compositions used in the experiments showed this feature. The first experimental procedures were designed to elucidate the equilibrium properties of the L1 and LLC phases before the dynamic properties relating the two phases were studied. The first studies consisted of dynamic light scattering performed on the micellar phase near the boundaries. The analysis of the light scattering data was made using the CONTIN program as implemented by Brookhaven Instruments and assuming a spherical shape.35 Table 1 presents the results of the dynamic light scattering (DLS) for two different assumptions. To convert the calculated diffusion coefficients of the micelles into a hydrodynamic size, the continuousphase viscosity is utilized in the Stokes-Einstein relationship.36 For the system under study, the first set of size calculations were made by assuming that the continuous-phase viscosity was the same as the “solvent” of the lecithin based on the phase behavior. The “solvent” is defined as the composition if all the lecithin is removed from the system. This produced a trend in which the hydrodynamic diameter of the micelles decreased with increasing surfactant concentration from a maximum of 23.6 nm to a minimum of 7.5 nm as reported under diameter 1 in Table 1. However, SXS in water at the concentrations studied has a viscosity which is highly dependent upon the sodium xylenesulfonate concentration. To determine a more appropriate value for the composition and, hence, the viscosity of the continuous phase, conductivity measurements were made. Figure 2 reports the variation in conductivity for the micellar phase as a function of lecithin concentration at different concentrations of SXS. As a first-order calculation, the reduction in conductivity with increased surfactant concentrations is divided between two factors. First, the geometric obstruction due to the creation of association structures reduces the conductivity. Second, the reduction in conductivity is due to the association of the hydrotrope (the conducting species) which decreases the number of free ions available. The first factor has been theoretically studied by Jo¨nsson et al.37 For the determination of the obstruction factor, as in the light scattering, a spherical (35) vanZanten, J. J. Chem. Phys. 1995, 102, 273. (36) Boon, J.P.; Yip, S. Molecular Hydrodynamics; McGraw-Hill: New York, 1980. (37) Jo¨nsson, B.; Wennerstro¨m, H.; Nilsson, P.; Linse, P. Colloid Polym. Sci. 1986, 264, 77.

Figure 3. Interlayer spacing for the LLC region plotted versus mole fraction of SXS assuming no water in the layer. The curve is a guide to the eye.

shape is chosen. The difference between the conductivity calculated based upon the geometric obstruction and the measured conductivity is taken as the measure of the amount of SXS associated. For the measured cases, the amount of association is calculated to be to first order 1 molecule of SXS to 1 molecule of lecithin which translates to approximately 45% by weight of hydrotrope is associated. Based upon the above calculations, the light scattering data are reanalyzed using a continuous phase solvent viscosity with an appropriately reduced quantity of SXS. The results are presented as diameter 2 in Table 1. There is no trend in micelle size with surfactant concentration. The absolute magnitude of the size is subject to the limitations in the computation of the viscosity and is no better than (10%. To investigate the lamellar liquid crystalline (LLC) region, small-angle X-ray scattering was used to determine the interlayer spacing (d). Figure 3 provides the results of the X-ray measurements over the entire LLC regime. From extrapolating to zero concentration, the thickness of the lipid layer per se is found for different sodium xylenesulfonate concentrations. The region of most interest in these investigations is for the compositions which are in equilibrium with the stopped-flow product. Based upon the tie lines seen in Figure 1, the region of interest is at less than 3% SXS. In this region, the interlayer spacing (d) is 4.67 ( 0.17 nm, which is a smaller spacing than that for the pure lecithin and water liquid crystals. The variation in the interlayer spacing as seen in Figure 3 based upon the mole fraction of SXS neglecting water shows two competing effects. The first effect is the decrease in interlayer spacing, which is attributed to an increase in entropy due to the interaction of the SXS with the lecithin. At higher sodium xylenesulfonate concentrations, an increase in interlayer spacing is seen. This is attributed to the movement of some of the ionized SXS into the aqueous layer of the liquid crystal. After elucidating the structural dimensions in the L1 and LLC phases, a series of stopped-flow experiments were performed. In these experiments, an initial solution (as indicated in the inset of Figure 1) is mixed under high shear with an equal volume of water to produce a final solution of which the compositions are also indicated in

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Figure 4. Response curves from the stopped-flow measurements based upon 90° light scattering based on sample A. The voltage is arbitrarily set.

Figure 5. Comparison of the trend for the present results (solid points) and previous investigations (dashed line).40

Figure 1. While the high shear rate destroys the structure initially present, 90° light scattering is used to monitor size change as a function of time. The observed response is due to the formation of amphiphilic association structures. Figure 4 presents a series of representative repetitions of the same stopped-flow experiment. The approximate shape of the response curve is that of a singleexponential decay. However, there are a series of random variations from a single-exponential decay in each run, which variability will be addressed later. The analysis, as reported in Table 2, gives the time constant of each decay which was computed by two different averaging methods agreeing within experimental variability. The first method was to take 15 or more individual stoppedflow experiments and average the value of the response for each measured time. This produces a smooth singleexponential curve and could be used as long as all experimental variables controlling the magnitude of the response were exactly the same. The other method is to fit each response curve with a single exponential and then average a large number of time constants. This method allows for day to day variations in the experimental setup. A series of experiments using both methods produced mean values with a variation of less than 5%. In addition, immediately after the stopped-flow procedure, the final solution sizes were measured by dynamic light scattering. The results are presented in Table 2. At these lower concentrations, the amount of SXS associated with the lecithin makes a negligible difference in the computed continuous-phase viscosity, which is used to determine the sizes of the vesicles that are produced. The final sizes determined increase with increasing surfactant concentration from a minimum diameter of 57 nm to a maximum of 502 nm. The size distribution of the vesicles is quite broad. To obtain a measure of the stability of the vesicles, the DLS experiments were repeated on the solutions until a change in the size and size distribution was seen. The change in the average size was significant such as in the case of sample D, Table 2, when the distribution went from 459 ( 71 to 943 ( 416 nm at the final measurement. The change in size as determined by DLS and as determined visually occurred at approximately the same time. Hence, the change from the “final” structure to a phase-separated solution was quite rapid once initiated but varied in time before onset based upon the concentration. Larger quantities of lecithin resulted in a significantly faster onset of separation than at low concentrations as seen in Table 2.

The final aspect of the analysis is to determine the basic mechanism of the formation of the vesicles. There are two apparent mechanisms, which may work individually or jointly to build up the vesicles. The first mechanism is the addition of monomer to monomer to form aggregates and the continued addition of monomers to grow the structure to the final size. The second additional mechanism is the combination of smaller aggregates to form the final structure. A total description of this joint mechanism as originally investigated by Aniansson28 and Kahlweit38 for micelles results in the equation:

1/τ ) (MXn/m + β0mX)/(1 + σ2X/m)

(1)

in which M is a constant independent of surfactant concentration, X is the ratio of associated to free surfactant molecules, σ is the width of the size distribution, m is the mean aggregation number, and β0 is a measurement of the disassociation constant assuming an entirely attractive potential. The equation may be generalized to vesicles with no alteration of the analysis. From the above analysis, two regimes may be studied via the current stopped-flow process in which concentration is the experimental variable. In the fragment addition regime, the time constant τ decreases with increasing surfactant concentration. Figure 5 plots the two regimes as found in the early micellar studies (dashed line)39,40 and the results of the present experiments (solid points). The results clearly show that the formation of the vesicles in this system is in the aggregation regime. In addition, there is confirming evidence from the stoppedflow measurement response curves. The likely cause of the variability of the decay curve from a single exponential can be attributed to the random addition of fragments to create structures that are growing at an approximately exponential rate but which exhibit statistical variations from the decay. The random nature of the variations is observed in that when averaging a series of experiments, the variations disappear. These results are not unexpected: the interfacial packing of the ionic surfactants provides a less dense structure due to the repulsion from the polar group charges. (38) Kahlweit, M. J. Colloid Interface Sci. 1982, 90, 92. (39) Zana, R. Polym. Mater. Sci. Eng. 1993, 69, 24. (40) Lessner, E.; Teubner, M.; Kahlweitz, M. J. Phys. Chem. 1981, 85, 3167.

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Conclusion The decay times for the formation phospholipid vesicles in conjunction with a hydrotrope were determined based upon the stopped-flow process. The variation in decay time as a function of concentration in conjunction with

Campbell et al.

the shape of the response curve demonstrates that lecithin/ sodium xylenesulfonate vesicles form via the collision of aggregates. LA9707742