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Langmuir 2001, 17, 6816-6821
Synthesis of pH-Degradable Nonionic Surfactants and Their Applications in Microemulsions Maithili Iyer,† Douglas G. Hayes,*,‡ and J. Milton Harris§ Department of Chemistry and Department of Chemical and Materials Engineering, University of Alabama in Huntsville, Huntsville, Alabama 35899, and Shearwater Polymers, Inc., 1112 Church Street, Huntsville, Alabama 35801 Received April 2, 2001. In Final Form: August 16, 2001 An oil-soluble pH-degradable nonionic surfactant with poly(ethylene glycol) monomethyl ether as the hydrophile and a cyclic ketal as the hydrophobe was synthesized for use in microemulsion-based protein extraction. The surfactant solubilized water in isooctane. Dynamic light-scattering measurements showed formation of fairly monodisperse water-in-oil microemulsions of radii 4-6 nm, with very strong intermicellar attractive interactions. The ternary phase diagram for the system surfactant/water/isooctane at 23 °C consists of one-, two-, and three-phase regions as well as gel-like phases. The well-known “fish” pattern occurred for the phase diagram of temperature vs surfactant concentration at a fixed ratio of water-to-oil (1/1 g/g). The surfactant remained stable at neutral pH for several days but degraded rapidly when a mildly acidic phosphate buffer (pH ) 5) was encapsulated in the water-in-oil microemulsion solution. Degradation occurred more rapidly when the microemulsion solution was brought in contact with an equal volume of pH 5 buffer solution in the presence of agitation. The encapsulation of protein (lysozyme) and its subsequent release upon contact with pH 5 buffer were observed, with 70% recovery of lysozyme mass in 0.5 h and 90% recovery in 1.0 h. The specific activity of the recovered lysozyme was within 90.4 ( 4.0% of the value for untreated lysozyme.
Introduction Water-in-oil microemulsions (w/o-µEs), nanometer-sized aqueous droplets dispersed in nonpolar solvents due to the action of surfactants, have been employed for enhanced oil recovery, the hosting of polymeric, organic, and enzymatic reactions, the refolding of proteins, and the separation of biomolecules.1-3 Water-in-oil microemulsionbased protein extraction (MPE) has been demonstrated to selectively isolate proteins and amino acids from complex fermentation broths.4-7 The “forward extraction” of protein into the microemulsion phase is a rapid process primarily driven by favorable interactions between the surfactant headgroup and the protein target (e.g., electrostatic, ion pairing, or bioaffinity) near the liquid-liquid interface. In contrast, the subsequent recovery or “back extraction” of encapsulated protein is often difficult, incomplete, and/or very slow.4-7 The primary approach for back extraction is to reduce the attraction between surfactant and encapsulated protein. For instance, a net positively charged protein extracted by an anionic surfactant containing microemulsion solution is back extracted by an aqueous stripping solution possessing a pH * To whom all correspondence should be addressed. † Department of Chemistry, University of Alabama in Huntsville. ‡ Department of Chemical and Materials Engineering, University of Alabama in Huntsville. § Shearwater Polymers, Inc. (1) Goto, M.; Hasimoto, Y.; Fujita, T.; Ono, T.; Furusaki, S. Biotechnol. Prog. 2000, 16, 1079. (2) Sjo¨blom, J.; Lindberg, R.; Friberg, S. E. Adv. Colloid Interface Sci. 1996, 95, 125-287. (3) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (4) Hayes, D. G. In Liquid Interfaces in Chemical, Biological, and Pharmaceutical Applications; Volkov, A., Ed.; Surfactant Science Series Vol. 95; Marcel Dekker: New York, 2001; p 469. (5) Pires, J. M.; Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Prog. 1996, 12, 290. (6) Leser, M. E.; Wei, G.; Luisi, P. L.; Maestro, M. Biochem. Biophys. Res. Commun. 1986, 135, 629. (7) Go¨klen, K. E.; Hatton, T. A. Biotechnol. Prog. 1985, 1, 69.
above the protein’s pI and high ionic strength. The latter reduces electrostatic attraction via Debye shielding of surfactant headgroups and reduces the interfacial curvature and hence the size and solubilizing capacity of the aqueous nanodroplets. Such an approach is often 1001000 times slower than forward extraction8 and is not successful for recovering biomolecules that strongly interact with the surfactant tail groups (e.g., via hydrophobic interactions).9,10 Several approaches have improved the rate and success of back extraction, especially for the release of surfactantinteractive proteins. One method is the inclusion of a polar cosolvent, such as ethanol, butanol, ethyl acetate, or 2-propanol, in the stripping solution to improve their recovery.9,10 However, use of polar solvents often leads to inactivation or unfolding of proteins.11-13 Other useful approaches include the dehydration of encapsulated water and the disruption of the interfacial packing of surfactant molecules by cosurfactants.4 An additional concern involving MPE is the frequent loss of the specific activity and stability of biomolecules encapsulated in ionic surfactant based w/o-µEs.3,4 Thus, the employment of nonionic surfactants or mixed nonionic/ ionic surfactant systems has gained popularity, since nonionic surfactants interact less strongly with biomolecules.14-17 Nonionic surfactants must be combined with (8) Dungan, S. R.; Bauch, T.; Hatton, T. A.; Plucinski, P.; Nitsch, W. J. Colloid Interface Sci. 1991, 145, 33. (9) Carlson, A.; Nagarajan, R. Biotechnol. Prog. 1992, 8, 85. (10) Woll, J. M.; Hatton, T. A.; Yarmush, M. L. Bioaffinity Separations Using Reversed Micellar Extraction. Biotechnol. Prog. 1989, 5, 57. (11) Mozhaev, V. V.; Khmelnitsky, Y. L.; Sergeeva, M. V.; Belova, A. B.; Klyachko, N. L.; Levashov, A. V.; Martinek, K. Eur. J. Biochem. 1989, 184, 597. (12) Ghatorae, A. S.; Guerra, M. J.; Bell, G.; Halling, P. J. Biotechnol. Bioeng. 1994, 44, 1355. (13) Toscano, G.; Pirozzi, D.; Greco, G., Jr. Biotechnol. Lett. 1990, 12, 821. (14) Shioi, A.; Harada, M.; Takahashi, M.; Adachi, M. Langmuir 1997, 13, 609.
10.1021/la010494t CCC: $20.00 © 2001 American Chemical Society Published on Web 10/02/2001
Synthesis of pH-Degradable Nonionic Surfactants
ionic or affinity surfactants to achieve significant extraction yields in most situations.14,16,17 The most successful nonionic surfactants for MPE were found to contain a hydrophilic-lipophilic balance, or HLB value, in the range of 8-11.16,17 The employment of surfactants with lower HLB values (i.e., that are more nonpolar) produced Winsor II systems (w/o-µE solution in equilibrium with an “excess” aqueous phase) that yielded poor disengagement of the two liquid phases.16 A further disadvantage of MPE is the limited capacity for solubilizing protein. The limitation is due to the use of low surfactant concentrations, since the separation of aqueous and organic phases is hindered by the presence of macroemulsions when the surfactant concentration is high. The use of cleavable or degradable nonionic surfactants for MPE may erase many of the above-mentioned disadvantages. The controlled degradation of cleavable surfactant will destroy the surface activity of the surfactants, hence disrupting w/o-µE (and macroemulsion) formation and leading to the release of encapsulated protein. Cleavable surfactants contain labile functional groups that form non-surface-active species or new surface-active compounds upon exposure to a specific physicochemical agent (acid, alkali, salt, heat, or light). Among the various types of known cleavable surfactants, compounds that decompose through the adjustment of pH are most common. Generally, alkaline-degradable surfactants contain a cleavable ester or Si-O bond and acid-degradable surfactants possess cyclic acetal, cyclic ketal, or ortho bonds.18-20 The use of pH-degradable surfactants in w/oµE has rarely been investigated.21 On the basis of previous work performed in our laboratory involving aqueous micellar surfactant systems (HLB values greater than 12),22 we have successfully synthesized a two-tailed oilsoluble nonionic surfactant using poly(ethylene glycol) monomethyl ether, MPEG-350, as the hydrophile and a cyclic ketal as the hydrophobe. The cyclic ketal linkages hydrolyzed under mildly acidic conditions (pH ∼ 5 or less), yielding two non-surface-active species, one of which resides in the aqueous phase (MPEG) and the other in the oil phase (ketone). Here, the synthesis procedure, the physical properties of surfactant/water/isooctane microemulsion systems, and the hydrolysis of surfactant and the solubilization and release of proteins from these systems are presented. Experimental Section Materials. MPEG-350, triethylamine (99.5% pure), mesyl chloride (99.5% pure), and p-toluenesulfonic acid (97% pure) were purchased from Aldrich, Milwaukee, WI. 3-Hexadecanone (97% pure) was purchased from Lancaster Synthesis, Windham, NH. Lysozyme from chicken egg white, 3 times crystallized, dialyzed, and lyophilized, containing approximately 95% protein with the balance being primarily buffer salts, N-acetylglucosamine (NAG, 99% pure), and glycol chitin (degree of polymerization ) 2500) (15) Ayala, G. A.; Kamat, S.; Beckman, E. J.; Russell, A. J. Biotechnol. Bioeng. 1992, 39, 806. (16) Vasudevan, M.; Tahan, K.; Wiencek, J. M. Biotechnol. Bioeng. 1995, 46, 99. (17) Vasudevan, M.; Wiencek, J. M. Ind. Eng. Chem. Res. 1996, 35, 1085. (18) Holmberg, K. In Novel Surfactants; Holmberg, K., Ed.; Surfactant Science Series Vol. 74; Marcel Dekker: New York, 1998; p 334. (19) Hellberg, P. E.; Bergstrom, K.; Holmberg, K. J. Surfactants Deterg. 2000, 3, 81. (20) Hellberg, P. E.; Bergstrom, K.; Juberg, M. J. Surfactants Deterg. 2000, 3, 369. (21) Steytler, D. C.; Sargeant, D. L.; Welsh, G. E.; Robinson, B. H.; Heenan, R. K. Langmuir 1996, 12, 5312. (22) Yue, C. Synthesis and Characterization of Cleavable Surfactants Derived from Poly(ethylene glycol) Monomethyl Ether. Ph.D. Thesis, University of Alabama in Huntsville, Huntsville, AL, 1996.
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Figure 1. Reaction scheme for surfactant synthesis. were purchased from Sigma, St. Louis, MO. Sodium acetate, sodium carbonate, and potassium ferricyanide were purchased from Fisher Scientific, Pittsburgh, PA. Deionized water was used throughout. All other chemicals were of high purity and were used without further purification. Surfactant Synthesis (Figure 1). A. Preparation of MPEG Mesylate (MPEGOSO2CH3). MPEG-350 (0.1 mol) dissolved in 400 mL of toluene was azeotropically distilled for 2 h under nitrogen. After the solution was cooled to room temperature, 100 mL of dry methylene chloride and 15.3 mL of triethylamine (distilled over calcium hydride) were added. After the mixture was cooled, 8 mL of mesyl chloride, previously distilled to improve optical clarity, was added dropwise. The mixture was then stirred overnight under nitrogen at room temperature. The resultant precipitate was filtered off, and the solvent was removed under reduced pressure. The residue was then dissolved in methylene chloride and washed with 0.1 M phosphate buffer (pH 7). The combined organic phase was dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The yield was 90-95%. B. Preparation of Cyclic Ketal. A mixture of 0.15 mol of glycerol, 0.07 mol of 3-hexadecanone, 1 g of p-toluenesulfonic acid, and 150 mL of toluene was azeotropically distilled until the accumulation of water into the Dean-Stark trap stopped as a result of the completion of the reaction. The reaction’s progress was also monitored using thin-layer chromatography. The resultant organic phase was washed with saturated aqueous sodium carbonate twice and then with distilled water and subsequently dried with magnesium sulfate. Finally, the solvent was removed by rotary evaporation. If a large amount of ketone was still present in the final product after the washings, further purification of the product was carried out using column chromatography. The yield was 80-85%. C. Preparation of Surfactant. Cyclic ketal (0.05 mol) was azeotropically distilled in 150 mL of toluene for 1 h. After the solution was cooled, 0.07 mol of NaH in mineral oil was added gradually, and the mixture was heated at 40 °C in an oil bath for 2 h. The mixture was then cooled to room temperature. MPEG mesylate (0.055 mol) was added to the mixture, and the mixture was refluxed overnight. The solvent was then removed by rotary evaporation. The residue was dissolved in water and extracted with ethyl acetate. After the solution was dried with anhydrous magnesium sulfate, ethyl acetate was removed under reduced pressure. The yield was 80-85%. The surfactant was 87% pure, with the major impurity being unreacted MPEG (liquid chromatographic analysis). For a few investigations, highly purified (96%) surfactant was employed. It was obtained by subjecting the 87% pure surfactant to chromatography on a low-pressure silica gel column, using mixtures of hexane and ethyl acetate to elute all materials. Chromatographic purification resulted in a low, 40-45%, recovery
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Figure 2. Phase diagram for water/surfactant/isooctane at 23 °C. L, L + W, L + W + O, and LR indicate an isotropic one-phase microemulsion solution (Winsor IV), microemulsion phase in equilibrium with excess water (Winsor II), microemulsion phase with excess water and oil (Winsor III), and a lamellar- or gelphase region, respectively. The phase boundary separating the two- and three-phase regions was not determined exactly and hence is denoted by a dashed curve in the figure. The circle, square, and triangle represent systems employed for analysis by DLS (Figure 4), surfactant hydrolysis in a Winsor IV system (Figure 5), and surfactant hydrolysis in a Winsor III system (Figure 6), respectively. of surfactant. The differences in phase behavior and physical properties between the highly purified and the nonpurified surfactant were very small in the primary system of interest, monophasic w/o-µE solutions of low surfactant concentration (
