Enhanced Stabilization of Vesicles by Compressed CO2 - Langmuir

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Langmuir 2009, 25, 196-202

Enhanced Stabilization of Vesicles by Compressed CO2 Wei Li, Jianling Zhang, Siqing Cheng, Buxing Han,* Chaoxing Zhang, Xiaoying Feng, and Yueju Zhao Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China ReceiVed September 25, 2008. ReVised Manuscript ReceiVed October 23, 2008 In this work, we studied the effect of compressed CO2 on the stability of vesicles formed in a dodecyltrimethylammonium bromide (DTAB)/sodium dodecyl sulfate (SDS) mixed surfactant system by combination of phase behavior and turbidity study, and UV-vis and fluorescence techniques. It was discovered that compressed CO2 could enhance the stability of vesicles significantly. This new and effective method to stabilize vesicles has some unique advantages over conventional methods. For example, the size and stability of the vesicles can be easily controlled by CO2 pressure; the method is greener because CO2 is a green reagent and it can be released completely after depressurization, which simplifies postseparation processes in applications. The main reason for CO2 to stabilize the vesicles is that CO2 molecules can insert into the hydrophobic bilayer region to enhance the rigidity of the vesicle film and reduce the size of the vesicles, which is different from that of conventional cosolvents (e.g., alcohols) used to stabilize vesicles. On the basis of this discovery, we developed a method to prepare hollow silica spheres using tetraethoxysilane as the precursor and CO2-stabilized vesicles as the template, in which CO2 acts as both the stabilizer of the vesicular template and the catalyst for the hydrolysis reaction of the precursor, and other cosolvents and catalysts are not required. Besides, the size of the silica hollow spheres prepared can be controlled by the pressure of CO2.

1. Introduction Vesicles in aqueous solutions can be defined as microscopic spherical bilayer structures containing an aqueous compartment enclosed by a lipid or surfactant bilayer. In the vesicle structure, the surfactant hydrophilic head groups are directed toward water, while the hydrophobic tails form the bilayer. Vesicular systems possess distinct microenvironments for the encapsulation of guest molecules. In aqueous solution, the entrapped aqueous interior provides discrete media for polar substrates and hydrophobic molecules can be incorporated into the lipid bilayer membranes. The special structure of vesicles makes them have a wide range applications in different fields, such as drug and gene delivery,1 material science,2 gelation,3 and chemical reaction.4 For a long time, vesicles had rarely formed without the input of considerable mechanical energy or elaborate chemical treatments, and specifically designed surfactants are usually required. In 1989, Kaler et al. gave the first example of spontaneous vesicle formation from mixed cationic and anionic single chain surfactants.5 Since then, the vesicular systems formed from mixtures of anionic and cationic surfactants in aqueous solutions have been extensively * To whom correspondence should be addressed. Telephone: +86-1062562821. Fax: +86-10-62559373. E-mail: [email protected]. (1) (a) Dias, R. S.; Lindman, B.; Miguel, M. G. J. Phys. Chem. B 2002, 106, 12600–12607. (b) Dias, R. S.; Lindman, B.; Miguel, M. G. J. Phys. Chem. B 2002, 106, 12608–12612. (2) (a) Hentze, H. P.; Raghavan, S. R.; McKelvey, C. A.; Kaler, E. W. Langmuir 2003, 19, 1069–1074. (b) Lootens, D.; Vautrin, C.; Van, H. D.; Zemb, T. J. Mater. Chem. 2003, 13, 2072–2074. (c) McKelvey, C. A.; Kaler, E. W.; Zasadzinski, J. A.; Coldren, B.; Jung, H. T. Langmuir 2000, 16, 8285–8290. (3) (a) Antunes, F. E.; Marques, E. F.; Gomes, R.; Thuresson, K.; Lindman, B.; Miguel, M. G. Langmuir 2004, 20, 4647–4656. (b) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. Macromolecules 1999, 32, 6626–6637. (c) Lee, J. H.; Gustin, J. P.; Chen, T. H.; Payne, G. F.; Raghavan, S. R. Langmuir 2005, 21, 26–33. (4) (a) Lasic, D. D. Liposomes: From Physics to Application; Elsevier: Amsterdam, 1993; Chapter 6. (b) Tadros, T. F. Applied Surfactant - Principle and Applications; Wiley-VCH: Weinheim, Germany, 2005; Chapter 3. (5) Kaler, E. W.; Murthy, A. K.; Rodriguez, B.; Zasadzinski, J. A. N. Science 1989, 245, 1371–1374.

studied.6 This kind of vesicular systems has some unique advantages. For example, simple and cheap surfactants can be used to prepare vesicles, and the preparation process is very simple. However, mixed cationic-anionic surfactant systems, especially 1:1 mixtures, usually precipitate in aqueous solutions due to partial shielding of charges.7 This limits the creation of vesicle systems and their applications greatly. To solve this problem, some additives such as organic cosolvents or cosurfactants have been used to stabilize vesicles.8 Supercritical or compressed CO2 has received much attention in recent years because it is readily available, inexpensive, nontoxic, and nonflammable. Compressed CO2 has been used in different fields, such as extraction and fractionation,9 chemical reactions and material science,10 controlling the stability of reverse micelles,11 induction of nanoemulsions,12 creating microemul(6) (a) Schnur, J. M. Science 1993, 262, 1669–1676. (b) Jain, S.; Bates, F. S. Science 2003, 300, 460–464. (c) Liu, T.; Diemann, E.; Li, H.; Dress, A. W. M. Nature 2003, 426, 59–62. (d) Yin, H. Q.; Zhou, Z. K.; Huang, J. B.; Zheng, R.; Zhang, Y. Y. Angew. Chem., Int. Ed. 2003, 42, 2188–2191. (e) Seredyuk, V. A.; Menger, F. M. J. Am. Chem. Soc. 2004, 126, 12256–12257. (f) Li, X.; Dong, S. L.; Jia, X. F.; Song, A. X.; Hao, J. C. Chem.sEur. J. 2007, 13, 9495–9502. (g) Sohrabi, B.; Gharibi, H.; Javadian, S.; Hashemianzadeh, M. J. Phys. Chem. B 2007, 111, 10069–10078. (h) Letizia, C.; Andreozzi, P.; Scipioni, A.; La Mesa, C.; Bonincontro, A.; Spigone, E. J. Phys. Chem. B 2007, 111, 898–908. (7) Christian, S. D.; Scamehorn, J. F. Solubilization in surfactant aggregates; Marcel Dekker, Inc.: New York, 1995; Chapter 4. (8) (a) Huang, J. B.; Zhu, B. Y.; Zhao, G. X.; Zhang, Z. Y. Langmuir 1997, 13, 5759–5761. (b) Feitosa, E.; Bonassi, N. M.; Loh, W. Langmuir 2006, 22, 4512–4517. (c) Brito, R. O.; Marques, E. F.; Gomes, P.; So1derman, O. J. Phys. Chem. B 2006, 110, 18158–18165. (9) Blanchard, L. A.; Hancu, D.; Bechman, E. J.; Brennecke, J. F. Nature 1999, 399, 28–29. (10) (a) Jessop, P. G.; Leitner, W. Chemical Synthesis Using Supercritical Fluids; Wiley-VCH: Weinheim, 1999; Chapter 5. (b) Poliakoff, M.; Fitzpatrick, J. M.; Farren, T. R.; Anastas, P. T. Science 2002, 297, 807. (c) Johnston, K. P.; Shah, P. S. Science 2004, 303, 482–483. (11) (a) Shen, D.; Zhang, R.; Han, B. X.; Dong, Y.; Wu, W.; Zhang, J. L.; Liu, J. C.; Jiang, T.; Liu, Z. M. Chem.sEur. J. 2004, 10, 5123–5128. (b) O’Callaghan, J. M.; Copley, M. P.; Hanrahan, J. P.; Morris, M. A.; Steytler, D. C.; Heenan, R. K.; Staudt, R.; Holmes, J. D. Langmuir 2008, 24, 6959–6964. (12) Zhang, J. L.; Han, B. X.; Zhang, C. X.; Li, W.; Feng, X. Y. Angew. Chem., Int. Ed. 2008, 47, 3012–3015.

10.1021/la8031545 CCC: $40.75  2009 American Chemical Society Published on Web 12/02/2008

Enhanced Stabilization of Vesicles

sions with CO2 as the continuous phase,13 changing the fluidity and melting point of liposome systems,14 and tuning the properties of organic solvents for different processes.15 It is known that addition of additives to surfactant solutions usually suffers from economic and environmental costs and makes the posttreatment much difficult in applications.16 Development of effective and greener methods to create and stabilize vesicles formed from cheap cationic-anionic surfactant mixtures is very interesting and challenging. In this work, we studied the effect of compressed CO2 on the stability of vesicles formed from two of the most commonly used surfactants, dodecyltrimethylammonium bromide (DTAB) and sodium dodecyl sulfate (SDS). Interestingly, it was discovered that compressed CO2 could enhance the stability of the vesicles significantly. This new method to stabilize vesicles has several advantages. For example, the stability and the size of the vesicles can be easily controlled by the pressure of CO2; the method is greener because CO2 is a green reagent, and it can be released completely after depressurization and can be easily reused, which can simplify postseparation processes in applications. The features of the CO2-stabilzed vesicular systems are favorable to their applications. As an example of application, we developed a method to prepare hollow silica spheres using tetraethoxysilane as the precursor and the CO2-stabilized vesicles as the template. In this method, CO2 acts as both the stabilizer of the vesicular template and the reagent to produce the acidic catalyst for the hydrolysis reaction of the precursor. This avoids the use of liquid acids and organic cosolvents, and the size of the silica hollow spheres can be controlled by CO2 pressure.

2. Experimental Section 2.1. Materials. DTAB (A.R. grade) and SDS (A.R. grade) were purchased from Shanghai Shanpu Chemical Corporation and Tianjin Jinke Fine Chemical Institute, respectively. They were recrystallized five times from ethanol-acetone mixed solvent before use. 2-(pToluidino)naphthalene-6-sulfonate (TNS, A.R. grade, Scheme S1 in the Supporting Information) and 6-propionyl-2-(dimethylamino)naphthalene (prodan, A.R. grade, Scheme S2 in the Supporting Information) used as the fluorescent probes were purchased from Sigma and Fluka, respectively. Dimethyl yellow (A.R. grade) and methyl orange (MO, A.R. grade) were produced by Beijing Chemical Reagent Factory. Tetraethoxysilane (TEOS, A.R. grade) was purchased from Tianjin Yongda Chemical Reagent Company. Sulfuric acid was provided by Beijing Chemical Plant. Citric acid (A.R. grade, Chengdu Chemical Plant) and trisodium citrate dihydrate (A.R. grade, Beijing Yili Fine Chemical Plant) were used to prepare the buffer solutions. CO2 (>99.995% purity) was provided by Beijing Analytical Instrument Factory. Double-distilled water was used throughout the experiments. 2.2. Effect of CO2 on the Phase Behavior of the Mixed Surfactant System. The apparatus and procedures to study the phase behavior of the mixed surfactant system were the same as those used previously.17 The apparatus consisted mainly of a view cell (34.0 mL) with a magnetic stirrer, a high-pressure pump (DB-80), a constant-temperature water bath, and a pressure gauge. The accuracy of the pressure gauge, which was composed of a transducer (FOXBORO/ICT, Model 93) and an indicator, was (0.025 MPa (13) (a) Hoefling, T. A.; Enick, R. M.; Beckman, E. J. J. Phys. Chem. 1991, 95, 7127–7129. (b) Beckman, E. J. Science 1996, 271, 613–614. (c) Johnston, K. P.; Harrison, K. L.; Clarke, M. J.; Howdle, S. M.; Heitz, M. P.; Bright, F. V.; Carlier, C.; Randolph, T. W. Science 1996, 271, 624–626. (d) Eastoe, J.; Gold, S.; Rogers, S.; Wyatt, P.; Steytler, D. C.; Gurgel, A.; Heenan, R. K.; Fan, X.; Beckman, E. J.; Enick, R. M. Angew. Chem., Int. Ed. 2006, 45, 3675–3677. (14) Geoffrey, D. B.; Barbara, L. K.; Herbert, J. S.; Sue, E. N. Langmuir 2005, 21, 530–536. (15) (a) Jessop, P. G.; Subramaniam, B. Chem. ReV. 2007, 107, 2666–2694. (b) Anand, M.; Odom, L. A.; Roberts, C. B. Langmuir 2007, 23, 7338–7343. (16) Liu, Y.; Jessop, P. G.; Cunningham, M.; Eckert, C. A.; Liotta, C. L. Science 2006, 313, 958–960. (17) Li, D.; Han, B. X. Macromolecules 2000, 33, 4555–4558.

Langmuir, Vol. 25, No. 1, 2009 197 within the pressure range of 0-20 MPa. The temperature of the water bath was controlled by using a HAAKE D8 temperature controller with an accuracy of (0.1 °C. In the experiment, the surfactant solutions of DTAB and SDS were first prepared separately. The two surfactant solutions of equal volume were then loaded into the view cell, and the stirrer was started. The view cell was placed in a water bath of 30.0 °C, and CO2 was charged slowly into the view cell to suitable pressure. After stirring for 30 min, the stirrer was stopped, and the time was recorded when the precipitation began, which was known by a change of the solution from clear to turbid. 2.3. UV-Vis Study. A UV-vis method was used to study the turbidity and micropolarity of the mixed surfactant system with and without CO2. The apparatus and procedures were similar to those reported previously.18 The apparatus consisted mainly of a gas cylinder, a high-pressure pump, a pressure gauge, a UV-vis spectrometer, and a temperature-controlled high-pressure UV sample cell. The UV-vis spectrophotometer was produced by Beijing General Instrument Company (model TU-1201) with a resolution of 0.1 nm. The sample cell was composed mainly of a stainless steel body, two quartz windows, a stirrer, and a temperature-controlling system. The optical path length and the inner volume of the cell were 2.1 cm and 8.8 mL, respectively. To determine the turbidity, the absorbance at the wavelength of 514.5 nm was monitored where no absorbance was observed for the mixed surfactant system.19 In the experiment, the sample cell was flushed with CO2 to remove the air in the cell. The two surfactant solutions of equal volume (Ctotal )10.0 mM) were charged into the sample cell of 30.0 °C, and the solution was stirred. CO2 was then compressed into the sample cell to desired pressure. The stirrer was stopped after 1 h, and the UV spectra of the solution were recorded at different times. The micropolarity of the vesicular system was studied using MO and dimethyl yellow as the probes. The procedures were similar to those used to study the turbidity discussed above. The main difference was that two surfactant solutions with a probe MO or dimethyl yellow were loaded into the sample cell. The concentrations of MO and dimethyl yellow in the solution were 2.5 and 0.2 µM, respectively. 2.4. Fluorescence Study. Steady-state fluorescence experiments were carried out with a HITACHI F-2500 fluorescence spectrophotometer, and the high-pressure fluorescence cell was similar to that used previously.20 TNS and prodan were used as the probes. Excitation wavelengths of 330 and 340 nm were chosen for TNS and prodan, respectively. The emission wavelengths for the two probes were varied from 340 and 350 to 600 nm, respectively. In all cases, the excitation and emission slit widths were kept at 2.5 nm and the scan rate was 60 nm/min. The experiment was also conducted at 30.0 °C. The final concentration of TNS and prodan in the surfactant solution were 10.0 and 5.0 µM, respectively. The spectra were analyzed by deconvolution into overlapping Gaussian curves using nonlinear least-squares-fitting method. An iterative Marquardt-Levenberg fitting algorithm21 was used to obtain the minimum number of reproducible absorbing components using the adjustable parameters of the center, width, and amplitude of each Gaussian curve. The percent area of each Gaussian curve was also calculated. The square of the multiple correlation coefficient, r2, was better than 0.999 in all the cases. 2.5. Preparation of Hollow Silica Spheres. The apparatus was similar to that used to study the effect of CO2 on the phase behavior of the mixed surfactant system, as was discussed above. In the experiment, 10.0 mL of DTAB/SDS vesicle solution (1:1, Ctotal ) 10 mM) was first prepared in the presence of CO2. Then 0.5 mL TEOS was added into the system and stirred for 24 h. After depressurization, (18) (a) Zhang, R.; Liu, J.; He, J.; Han, B. X. Macromolecules 2002, 35, 7869–7871. (b) Lu, J.; Han, B. X.; Yan, H. K. Phys. Chem. Chem. Phys. 1999, 1, 3269–3276. (19) (a) Yin, H. Q.; Huang, J. B.; Lin, Y. Y.; Zhang, Y. Y.; Qiu, S. C.; Ye, J. P. J. Phys. Chem. B 2005, 109, 4104–4110. (b) Yin, H. Q.; Huang, J. B.; Gao, Y. Q.; Fu, H. L. Langmuir 2005, 21, 2656–2659. (20) Liu, D. X.; Zhang, J. L.; Fan, J. F.; Han, B. X.; Chen, J. J. Phys. Chem. B 2004, 108, 2851–2856. (21) (a) Marquardt, D. W. J. Soc. Ind. Appl. Math. 1963, 11, 431–441. (b) Levenberg, K. Q. Appl. Math. 1944, 2, 164–168.

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Figure 1. Dependence of phase separation time on CO2 pressure at 30.0 °C for SDS/DTAB aqueous solution (1:1, Ctotal ) 10.0 mM).

the upper white latex was collected and washed with water and ethanol several times. The experiment without CO2 was also conducted, and the procedures were similar to those described above. The main differences were that a little amount of sulfuric acid was used as the catalyst for the hydrolysis reaction and the pH of the solution was 3.0. The morphology of the obtained particles was characterized by transmission electron microscopy (TEM) with a TECNAI 20 PHILIPS electron microscope. In the experiment, the particles were dispersed in ethanol and then directly deposited on the copper grid. X-ray diffraction (XRD) analysis of the samples was performed on a diffractometer (model D/MAX2500, Rigaka) with Cu KR radiation at a scanning rate of 2 min-1. The IR spectra of the samples were recorded using a Fourier transform infrared (FT-IR) spectrometer (Bruker TENSOR 27) with 32 scans at an effective resolution of 4 cm-1. Samples were grounded with KBr and pressed into thin and transparent wafers. X-ray photoelectron spectroscopy (XPS) charaterization was performed by means of an ESC ALab220i-XL spectrometer at a pressure of about 3 × 10-9 mbar using Al KR as the exciting source (hν )1486.6 eV) and operated at 15 kV and 20 mA.

3. Results and Discussion 3.1. Phase Behavior in the Presence of CO2. DTAB and SDS precipitated after their solutions (1:1, Ctotal ) 10.0 mM) were mixed for enough time. The concentration was much larger than the cvc (0.001 wt%) of the mixed system.22 Figure 1 shows the time required for phase separation to occur after mixing. It can be seen that addition of CO2 to the system could prolong the time effectively. In the absence of CO2, phase separation occurred immediately after the two surfactant solutions were mixed, which was known from the fact that the solution became turbid. However, the system could keep one phase for 15 days at CO2 pressure of 6.00 MPa. 3.2. Turbidity of the Solution. UV-vis spectroscopy is often used to study the turbidity of the solutions. 23 In this work, we studied the turbidity of the solution using this method. Figure 2 shows the dependence of turbidity with and without CO2 on time. In the absence of CO2, the precipitation of the surfactants occurred quickly after the solutions of the two surfactants were mixed. The absorbance changed from 0.6 to 1.6 in 5 h, as can be known from Figure 2a. The turbidity then decreased with increasing time because precipitated surfactants deposited at the bottom of the sample cell gradually, which reduced the amount of the surfactants in the solution. This process can also be seen clearly from the photos in Figure 2b (top row). Precipitate could be observed at the bottom of the glass bottle after 6 h. Figure 2a also shows that in the presence of compressed CO2 the turbidity changed slightly with time at the beginning and then kept constant for a long time. The photographs in Figure 2b (bottom row)

show that the original transparent solution turned slightly bluish. After that, there was no distinct change with time, indicating that the compressed CO2 could enhance the stability of the mixed surfactant solution. The turbidity change with time is consistent with the direct observation. 3.3. Micropolarity. A UV-vis method was used to characterize the vesicles formed in the DTAB/SDS mixed surfactant system, and hydrophobic dimethyl yellow was used as the probe, since it has less effect on the electrostatic interaction in cationic-anionic surfactant systems. The method was similar to that reported in the literature.24 It is well-known that the UV spectra of the chromophore are related to the polarity of the media.25 As examples, Figure 3 illustrates the UV spectra of the probe in the DTAB/SDS mixed surfactant system and in water with and without CO2. The absorption maximum (λmax) of dimethyl yellow in pure water appeared at 456 and 500 nm. The λmax in the surfactant solution appeared at 415 nm without CO2 and shifted to 408 nm in the presence of CO2 at 6.04 MPa. The obvious blue shift of λmax indicates that the polarity of the environment of the dye is weaker after adding CO2. It can also be known from spectrum 2 in Figure 3 that there is obvious absorption at about 456 nm in the absence of CO2, while there

(22) Herrington, K. L.; Kaler, E. W. J. Phys. Chem. 1993, 97, 13792–13802. (23) (a) Wang, C. Z.; Wang, S. Z.; Huang, J. B.; Li, Z. C.; Gao, Q.; Zhu, B. Y. Langmuir 2003, 19, 7676–7678. (b) Rosa, M.; Infante, M. R.; Miguel, M. D. G.; Lindman, B. Langmuir 2006, 22, 5588–5596.

(24) Yan, Y.; Huang, J. B.; Li, Z. C.; Han, F.; Ma, J. M. Langmuir 2003, 19, 972–974. (25) Division of Instrument Analysis of Chemistry Department. The Tutorial of Instrument Analysis; Peking University Press: Beijing, 1997; p 22.

Figure 2. Dependence of turbidity of DTAB/SDS system (1:1, Ctotal )10.0 mM) on time with and without CO2: (a) UV absorbance and (b) photographs without CO2 (top row) and with CO2 at 6.04 MPa (bottom row).

Enhanced Stabilization of Vesicles

Figure 3. UV-vis spectra of dimethyl yellow in different systems: (1) water without CO2; (2) DTAB/SDS solution (1:1, Ctotal ) 10.0 mM) without CO2; and (3) DTAB/SDS solution (1:1, Ctotal ) 10 mM) with CO2 at 6.04 MPa. The results were obtained after the two surfactant solutions were mixed for 10 min.

Figure 4. UV-vis spectra of MO in mixed surfactant solutions: (1) DTAB/ SDS solution (1:1, Ctotal ) 10.0 mM) without CO2; (2) DTAB/SDS solution (1:1, Ctotal ) 10.0 mM) with CO2 at 6.04 MPa; (3) pure water without CO2; and (4) pure water with CO2 at 6.04 MPa. The results were obtained after the two surfactant solutions were mixed for 10 min.

is no absorption at this wavelength in the presence of CO2. A reasonable explanation for the larger λmax and wide absorption range in the absence of CO2 is that the bilayer membrane of the vesicles is hydrophobic. In the absence of CO2, the stability of the vesicles is poor and there are less vesicles. Therefore, the probe distributes between the membrane, interfacial region, and bulk water. However, in the presence of CO2, the vesicles are more stable and the probe exists in the membrane of the vesicles. The λmax of MO is also sensitive to the polarity of its environment, and the λmax shifts to longer wavelength as the polarity increases. It is another commonly used probe.26 MO was also employed as probe to further characterize the vesicular system. Figure 4 shows the UV spectra of MO with and without CO2. When CO2 exists in the system, the λmax was at 410 nm (spectrum 2), and there is obvious absorption at 500 nm, which corresponds to the absorption of MO in water in the presence of CO2 (spectrum 4). However, the λmax was at 415 nm (spectrum 1) in the absence of CO2, and there was no other absorption peak. This can also be explained by the fact that, in the absence of CO2, the stability of the vesicles was poor and the surfactants existed mainly in the forms of vesicles and suspended precipitates because the cvc is very small and there is no monomer. The surfactant molecules in the suspended precipitates may be not as well ordered as those in the vesicles. Therefore, more water can enter the region where MO existed. However, in the presence of CO2, more surfactant existed in the form of vesicles, which is more ordered, and the polarity of the membrane/water interface region where MO is located is weaker. Therefore, λmax is smaller and some MO (26) (a) Zhu, D. M.; Schelly, Z. A. Langmuir 1992, 8, 48–50. (b) Clarke, M. J.; Harrison, K. L.; Johnson, K. P.; Howdle, S. M. J. Am. Chem. Soc. 1997, 119, 6399–6406.

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Figure 5. Dependence of fluorescence intensity of TNS in DTAB/SDS aqueous solution (1:1, Ctotal ) 10.0 mM) on time and pressure.

molecules exist in the aqueous phase because MO is more hydrophilic than dimethyl yellow. 3.4. Fluorescence Study. Steady-state fluorescence spectroscopy experiments were carried out to characterize the vesicular system further. TNS and prodan were used as the probes. The use of TNS or prodan is very advantageous in analyzing the surface and interior of mixed aggregates constituted by cationic and anionic components.27 Both emit weakly in water but exhibit intense fluorescence upon binding to the membrane surface.28 A shift in the wavelength of maximum emission accompanies the enhancement of TNS fluorescence with a decrease in solvent polarity. TNS emits maximally at 420-450 nm in organic solvents of low polarity and weakly at about 465 nm in water.28 Hydrophobic interactions between TNS and surfactant would locate TNS in the hydrophobic environment and enhance the fluorescence quantum yield.29 In the vicinity of the aggregates with little or no net surface charge, the polycyclic aromatic TNS might also be expected to approach the hydrophobic regions and interact at the polar-nonpolar water-surfactant interface.30 Vesicle concentration was the main factor to govern the intensity of the peak (Imax).27 We examined the spectra of TNS fluorescence in the DTAB/SDS system. Figures 5 shows the dependence of fluorescence intensity of TNS in DTAB/SDS aqueous solutions (1:1, Ctotal ) 10.0 mM) on time at different pressures. The TNS and prodan-doped vesicles usually need several hours to stabilize the intensity on the fluorescence signal. Therefore, the reason for the change of intensity at the beginning is complex. The intensity at longer time gives the information of stability of the vesicles. It is clear that without CO2 the concentration of vesicles in the system decreased quickly with time due to the precipitation of the surfactants, and the intensity decreased from about 130 to 10 in 52 h. However, the intensity of the peak changed much slowly in the presence of CO2. This implied that the stability of the vesicles formed in the solution was enhanced significantly by CO2. The intensity could keep nearly at a fixed value for a long time in the presence of CO2, and the fixed value increased with the increase of CO2 pressure. This indicates that most of (27) (a) Kerry, K. K.; Candace, A. Z.; Marja, J. F. Langmuir 2003, 19, 10054– 10060. (b) Aicart, E.; Burgo, P. d.; Llorca, O.; Junquera, E. Langmuir 2006, 22, 4027–4036. (c) Junquera, E.; Burgo, P. d.; Boskovic, J.; Aicart, E. Langmuir 2005, 21, 7143–7152. (28) (a) Minardi, R. M.; Schulz, P. C.; Vuano, B. Colloids Surf., A 2002, 197, 167–172. (b) Weber, G.; Laurence, D. J. R. Proc. Biochem. J. 1954, 56, 31. (c) McClure, W. O.; Edelman, G. M. Biochemistry 1966, 5, 1908–1919. (d) McClure, W. O.; Edelman, G. M. Biochemistry 1967, 6, 567–572. (e) McClure, W. O.; Edelman, G. M. Acc. Chem. Res. 1968, 1, 65–70. (29) Karukstis, K. K. Handbook of Surfaces and Interfaces of Materials; Academic Press: San Diego, 2001; p 54. (30) (a) Chiang, H. C.; Lukton, A. J. Phys. Chem. 1975, 79, 1935–1939. (b) Flanagan, M. T.; Ainsworth, S. Biochim. Biophys. Acta 1968, 168, 16–26. (c) Lesslauer, W.; Cain, J. E.; Blasie, J. K. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 1499–1503. (d) Krishnan, K. S.; Balaram, P. Arch. Biochem. Biophys. 1976, 174, 420–430. (e) Cheng, S.; McQueen, H. M.; Levy, D. Arch. Biochem. Biophys. 1978, 189, 336–343.

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Figure 7. Dependence of fluorescence intensity of prodan in DTAB/ SDS aqueous solution (1:1, Ctotal ) 10.0 mM) on time and pressure.

Figure 6. (a) Resolution of TNS emission spectrum for DTAB/SDS aqueous solution (1:1, Ctotal ) 10.0 mM) at CO2 pressure of 6.04 MPa. (b) Effect of CO2 pressure on area percent of Gaussian peaks of TNS fluorescence emission in the DTAB/SDS aqueous solution (1:1, Ctotal ) 10.0 mM; A1, area% at 422 nm; A2, area% at 445 nm; A3, area% at 476 nm). The results were obtained after mixing the two solutions for 10 h.

the vesicles could exist for a long time in the presence of CO2, which is consistent with the conclusion obtained from Figure 2. To investigate the fluorescence emission results further, deconvolution of the spectra was conducted in this study. Three Gaussian curves of varying contribution were yielded in the deconvolution of the TNS spectra, and Figure 6a gives results of the spectrum after mixing the two solutions for 10 h at 6.04 MPa. The Gaussian components of the band of the vesicular system are centered at 422 nm (A1), 445 nm (A2), and 476 nm (A3). These peaks can be assigned to the following microenvironments: (1) emission at about 422 nm is attributed to TNS inside the hydrophobic bilayer of vesicles; (2) emission at 445 nm is assigned to TNS in the bulk solvent; and (3) emission at around 476 nm is assigned to the TNS at the vesicle surface.27 Figure 6b shows the area percents of the bands centered at 422 nm (A1), 445 nm (A2), and 476 nm (A3), which were obtained at different pressures after mixing the two solutions for 10 h. It can be known that (A1 + A3) is less than A2 without CO2, while (A1 + A3) is larger than A2 in the presence of CO2, indicating that the probe, TNS, is predominantly solubilized within the vesicles in the presence of CO2. The figure also illustrates that with increasing CO2 pressure the percentage contribution of A1 and A3 increases, while the percentage contribution of A2 decreases. This indicates that more TNS molecules exist in the vesicle surface and hydrophobic bilayer at higher pressure. The main reason is that the concentration of vesicles increased with increasing CO2 pressure, as discussed above. In other words, CO2 stabilized vesicles effectively and the ability increases with increasing pressure. The soluble nature of prodan in a wide range of solvents enables its distribution into an array of single-phase and multiphasic

regions. 31 Furthermore, the fluorescence signal can be used to characterize the properties of vesicular systems due to the sensitivity of the wavelength of maximum emission (λmax) to the polarity of prodan’s environment. 31 Deconvolution of the prodan fluorescence emission spectrum into a sum of overlapping Gaussian curves can provide information about the location of prodan within discrete microdomains of different polarities. In this work, we also examined the prodan fluorescence spectra of 5.0 µM in the DTAB/SDS vesicular system at different conditions. Figure 7 demonstrates the dependence of the fluorescence intensity of prodan in DTAB/SDS aqueous solution (Ctotal ) 10.0 mM) with time at different pressures. It is shown that the fluorescence intensity increased with increasing pressure. The fixed value could keep for a long time in the presence of CO2, while it decreased rapidly in the absence of CO2. The fixed value increased with the increase of pressure. This further indicates that CO2 can enhance the stability of the vesicles effectively. The deconvolution of prodan spectra was also conducted, and three Gaussian curves were yielded. As an example, Figure 8a shows deconvolution results of the spectrum obtained at 6.04 MPa after the two solutions were mixed for 10 h. The Gaussian components of the band are centered 470, 510, and 576 nm. These peaks can be assigned to the following microenvironments: (1) the emission at 470 nm is attributed to the prodan inside the hydrophobic bilayer; (2) the emission at 510 nm comes from prodan at the aggregate/water interface; and (3) the 576 nm emission can be assigned to prodan in the bulk solvent.28 Figure 8b presents the effect of pressure on the area percents of the Gaussian components centered at 470 nm (B1), 510 nm (B2), and 576 nm (B3). The spectra were also determined after mixing the two solutions for 10 h. The deconvolution parameters are presented in the Supporting Information (Table S2). It can be seen from the figure that prodan predominantly solubilized within the vesicles because (B1 + B2) is much larger than B3. As pressure is lower than about 2.50 MPa, the prodan molecules at the vesicle/water interface were more than that in the hydrophobic membrane, as is evidenced by B1 < B2. At higher pressure, however, more probe existed in the hydrophobic membrane due to B1 > B2. This also shows that CO2 of higher pressure is more effective for stabilizing the vesicles. 3.5. Preparation of Hollow Silica Particles Using the Vesicular Template. Vesicles are commonly used as templates (31) (a) Karukstis, K. K.; Kao, M. Y.; Savin, D. A.; Bittker, R. A.; Kaphengst, K. J.; Emetarom, C. M.; Naito, N. R.; Takamoto, D. Y. J. Phys. Chem. 1995, 99, 4339–4346. (b) Karukstis, K. K.; Suljak, S. W.; Waller, P. J.; Whiles, J. A.; Thompson, E. H. Z. J. Phys. Chem. 1996, 100, 11125–11132. (c) Karukstis, K. K.; Frazier, A. A.; Martula, D. S.; Whiles, J. A. J. Phys. Chem. 1996, 100, 11133–11138.

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Figure 8. (a) Resolution of prodan emission spectrum for DTAB/SDS aqueous solution (1:1, Ctotal ) 10.0 mM). (b) Effect of CO2 pressure on area % of the Gaussian peaks of prodan fluorescence emission in DTAB/ SDS aqueous solution (1:1, Ctotal ) 10.0 mM; B1, area % at 470 nm; B2, area % at 510 nm; B3, area % at 576 nm). The results were obtained after mixing the two solutions for 10 h.

to synthesize various kinds of materials.32 It is well-known that TEOS can hydrolyze to form silica under acidic conditions.33 It is known that an aqueous solution becomes acidic after dissolving CO2 and returns to neutral after releasing CO2. As discussed above, CO2 can also stabilize vesicles formed from DTAB/SDS. On the basis of the above study, we proposed a new method to prepare hollow silica particles, in which CO2 acts both as the stabilizer of the template and catalyst, and the TEM images are shown in Figure 9. As shown in Figure 9a-d, hollow silica particles were formed, and the size of the hollow spheres prepared at 3.50 MPa is larger than that of spheres fabricated at 6.02 MPa. It was estimated from TEM images that the particle size obtained at 3.50 MPa is about 600 nm and that of particles prepared at 6.02 MPa is about 200 nm. This indicates that the particle size can be tuned by changing the pressure of CO2. In addition, it can be deduced that the vesicles formed at higher pressure were smaller because the size of the silica particles reflected that of the template. Silica particles were also prepared in the absence of CO2 using sulfuric acid as the catalyst at pH ) 3.0, and the TEM image is presented in Figure 9e. The figure shows that large aggregates formed. The main reason may be that the vesicles were not stable in the absence of CO2. This demonstrates that CO2 acts as both the stabilizer of the vesicular template and the catalyst for the hydrolysis reaction of the precursor. The silica particles obtained were also characterized by XPS, FT-IR, and XRD techniques, which further confirmed that silica particles were formed (Supporting Information Figure S1). (32) (a) Zhang, X. J.; Li, D. Angew. Chem., Int. Ed. 2006, 45, 5971–5974. (b) Tanev, P. T.; Pinnavaia, T. J. Science 1996, 271, 1267–1269. (c) Tan, B.; Lehmler, H. J.; Vyas, S. M.; Knutson, B. L.; Rankin, S. E. AdV. Mater. 2005, 17, 2368– 2371. (33) Fidalgo, A.; Ilharco, L. M. Chem.sEur. J. 2004, 10, 392–398.

Figure 9. TEM images of silica particles prepared in DTAB/SDS aqueous solution (1:1, Ctotal ) 10.0 mM) at 3.50 MPa (a,b), 6.02 MPa (c,d), and without CO2 using sulfuric acid (pH ) 3.0) as the catalyst (e).

3.6. Discussion of Mechanism. It is well-known that CO2 can dissolve in water and can be ionized partially (H2O + CO2 ) H+ + HCO3-), which makes the water acidic, and the pH can be reduced to 3.2 at 6.0 MPa. 34 In order to clarify the effect of pH value on the stability of the vesicles, we used citric acid buffers to fix the pH values of DTAB/SDS solution in the absence of CO2, and the pH values of the solution were 3.0, 4.0, 5.0, and 6.0. It was demonstrated that precipitation of the surfactants occurred immediately after the solutions of the two surfactants were mixed, which was similar to the case of the neutral solution. This suggests that the change of acidity caused by CO2 was not the reason for the stabilization of the vesicles. Generally, the formation of vesicles from mixed surfactants can be explained by the geometry rule. According to this rule, the critical molecular packing parameter is given by P ) V/a0lc, where V and lc are the volume and chain length of the hydrophobic group, respectively, and a0 is the optimum area per polar group. For vesicle formation, the proper value of P falls in the range of 0.5-1, while for micelle formation P is usually less than 0.5, and precipitation (34) Liu, D. X.; Zhang, J. L.; Han, B. X.; Fan, J. F.; Mu, T. C.; Liu, Z. M.; Wu, W. Z.; Chen, J. J. Chem. Phys. 2003, 119, 4873–4878.

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Figure 10. Illustration of the possible mechanism to enhance the stability of DTAB/SDS vesicles by compressed CO2. The vesicles are first formed; then most surfactants precipitate in the absence of CO2 due to poor stability; and CO2 inserts into the bilayer region of the vesicles to reduce the size of the vesicles and enhance the rigidity of the bilayer membrane.

Li et al.

also enhances stability of the vesicles; and third, an increase in curvature of the outside layer of the membrane should reduce the stability of the vesicles because the gap between the surfactant molecules becomes larger. The three factors compete, and the two favorable factors are dominant. Moreover, insertion of CO2 into the outside layer may reduce the unfavorable effect. Therefore, the vesicles are stable in the presence of CO2. It should be emphasized that the mechanism for the interesting phenomenon needs to be studied further. It has been reported that conventional cosolvents, such as alcohols, stabilize vesicles by residing in the polar group layer36 and changing the dielectric constant of the solvent,28 because the cosolvents have polar groups. While CO2 is a nonpolar compound, and exists preferably in the hydrophobic region, this suggests that the mechanism to stabilize the vesicles by CO2 is different from that of conventional cosolvents.

4. Conclusion occurs when the P value is larger than 1.35 Before mixing, the P value of the two surfactant solutions was less than 0.5 because micelles are formed in the solutions. After two solutions were mixed, a0 decreases due to the interaction of the anionic and cationic surfactants. V and lc are almost unchanged. So, the P value increases to greater than 1, and the precipitates are formed. Besides the P value, some other factors also affect the formation and stability of vesicles, such as the stability and rigidity of the membrane, size of the vesicles, and counterion binding.19 In other words, even if the P value is the range of 0.5-1, vesicles may not be formed or may be unstable, depending also on other factors. Study on the mechanism for stabilization of the vesicles by CO2 is very interesting and challenging. At present, it is very difficult for us to give an exact mechanism on this. However, we can discuss some possible reasons on the basis of the experimental results of this work and some well-known knowledge. As discussed above, the effect of CO2 on properties of the bulk aqueous phase is not considerable for the stabilization of the vesicles. It was reported that the compressed CO2 can insert into surfactant tail region of Triton X-100/cyclohexane reverse micelles, which affects the stability of the reverse micelles.11a CO2 can also accumulate in the hydrophobic region of liposome bilayers and change the fluidity and melting point of the liposome system.14 It was also reported that CO2 can insert into the hydrophobic region of micelles to swell the micelles.11b It is known that CO2 is nonpolar and it is very soluble in alkanes and other organic liquids.15 Therefore, it can be deduced that the enhanced stability mainly results from the effect of CO2 on the properties of the vesicle membrane. One of the possible reasons is that CO2 can insert into the membrane to increase the rigidity of the membrane and reduce the size of the vesicles. This is schematically shown in Figure 10 and is explained in the following. Geometrically, DTAB and SDS can form vesicles when the mixing ratio is equimolar.19 However, in the absence of CO2, the vesicles are not stable and the surfactants precipitate. As discussed above, the diameter of the vesicles is reduced by addition of CO2. This indicates that CO2 can insert into the hydrophobic region of the bilayer membrane of the vesicles because the curvature of the bilayer membrane is increased. This is understandable because CO2 is a small nonpolar molecule and is easy to insert into the membrane region. Insertion of CO2 into the membrane affects the stability of vesicles mainly in three ways. First, rigidity of the vesicle membrane is enhanced, which is favorable to stabilizing the vesicles; second, the vesicles become smaller and the vesicles are more rigid, which

Development of effective and greener methods to create and stabilize vesicles is very interesting and challenging. We have studied the effect of compressed CO2 on the stability of vesicles formed in the DTAB/SDS (1:1) mixed surfactant system and application of the CO2-stabilzed vesicles in the synthesis of silica hollow spheres, and the main conclusions are as follows. Compressed CO2 can enhance the stability of vesicles effectively. The surfactants precipitate immediately after mixing of the solutions of the two surfactants without CO2. In the presence of CO2, however, vesicles can be stable for 15 days. This new method to stabilize vesicles has some unique advantages, such as the size and stability of the vesicles can be easily controlled by CO2 pressure, the method is greener, and CO2 can be released completely after depressurization, which simplified postseparation processes in applications. It is demonstrated that the change of acidity of an aqueous solution caused by addition of CO2 is not the main reason for the stabilization of the vesicles. Therefore, it can be deduced that the effect of CO2 on the properties of the vesicle membrane is the main reason. One of the main reasons for the enhanced stability of the vesicles is that CO2 can insert into the bilayer membrane of the vesicles, which enhances the rigidity of the vesicle film and reduces the size of the vesicles. This mechanism is different from that of conventional cosolvents (e.g., alcohols) used to stabilize vesicles. Hollow silica spheres can be prepared using tetraethoxysilane as the precursor and CO2-stabilized vesicles as the template. In this route, liquid cosolvents and liquids acid are not required because CO2 acts as both the stabilizer of the vesicular template and the reagent to produce the acidic catalyst for the hydrolysis reaction of the precursor. The size of the silica hollow spheres decreases with increasing CO2 pressure. We believe that this greener method to stabilize vesicles has potential application in some fields, such as materials science and chemical reaction. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20633080) and Chinese Academy of Sciences (KJCX2.YW.H16). Supporting Information Available: Chemical structure of

(35) (a) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1991; Chapter 17. (b) Yu, W. Y.; Yang, Y. M.; Chang, C. H. Langmuir 2005, 21, 6185–6193.

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fluorescence probe TNS and prodan, the XPS spectrum, IR spectrum, and XRD patterns of the silica particles prepared in the DTAB/SDS aqueous solution at 6.02 MPa, and parameters of the deconvoluted Gaussian components of the TNS and prodan fluorescence emission spectra at different pressures. This material is available free of charge via the Internet at http://pubs.acs.org.

(36) Svenson, S. Curr. Opin. Colloid Interface Sci. 2004, 9, 201–212.