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Stabilization of r-Chymotrypsin and Lysozyme Entrapped in Water-in-Silicone Oil Emulsions Paul M. Zelisko and Michael A. Brook* Department of Chemistry, McMaster University, Hamilton, Ontario, Canada L8S 4M1 Received April 25, 2002. In Final Form: August 1, 2002 A system of entrapping proteinaceous material in water-in-silicone oil emulsions using silicone surfactants is described. Although contact between cyclic and linear silicones and proteins is well-known to facilitate protein/enzyme denaturation, the additional presence of silicones bearing a few hydrophilic groups (silicone surfactants) in these W/O systems dramatically lowers the degree of protein denaturation. Spectrophotometric activity assays of R-chymotrypsin and lysozyme performed using a UV/visible spectrophotometer revealed that the enzymes entrapped within these emulsion systems retain activity that is equal to, or in some cases greater than, that of controls. The results suggest that these emulsions are a viable means by which to store proteinaceous materials.
Introduction The behavior of proteins at interfaces has garnered a great deal of attention because, among other things, of the importance of such interactions in biological systems. Proteins have been shown to adsorb or interact at solid/ liquid and solid/air interfaces1-17 as well as to play an integral role in the formation and stabilization of many emulsion (liquid/liquid) systems.18-28 As a result, a great deal is known about the behavior of proteins in contact with hydrocarbons and hydrocarbon-based surfactants.29,30 * To whom correspondence should be addressed. Phone: +1 (905) 525-9140 ext. 23483. Fax: +1 (905) 522-2509. (1) Gunning, P. A.; Mackie, A. R.; Kirby, A. R.; Morris, V. J. Langmuir 2001, 17, 2013. (2) Valstar, A.; Almgren, M.; Brown, W. Langmuir 2000, 16, 922. (3) Vasilescu, M.; Angelescu, D. Langmuir 1999, 15, 2635. (4) Lu, J. R.; Su, T. J. Langmuir 1998, 14, 6261. (5) Amado, F. M. L.; Santana-Marques, M. G.; Ferrer-Correia, A. J.; Tomer, K. B. Anal. Chem. 1997, 69, 1102. (6) Beaglehole, D.; Lawson, F.; Harper, G.; Hossain, M. J. Colloid Interface Sci. 1997, 192, 266. (7) Duncan, A. C.; Sefton, M. V.; Brash, J. L. Biomaterials 1997, 18, 1585. (8) Xia, J.; Qian, J.; Nnanna, I. A. J. Agric. Food Chem. 1996, 44, 975. (9) Okubo, M.; Ahmad, H. Colloid Polym. Sci. 1996, 274, 112. (10) Wang, R.; Sun, S.; Bekos, E. J.; Bright, F. V. Anal. Chem. 1995, 67, 149. (11) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (12) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (13) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159. (14) Tan, J. S.; Martic, P. A. J. Colloid Interface Sci. 1990, 136, 415. (15) Mizutani, T.; Brash, J. L. Chem. Pharm. Bull. 1988, 36, 2711. (16) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988, 124, 28. (17) Norde, W.; MacRitchie, F.; Nowicka, G.; Lyklema, J. J. Colloid Interface Sci. 1986, 112, 447. (18) Chang, J. H.; Lee, S. C.; Lee, W. K. Chem. Eng. J. 1999, 73, 43. (19) Dickinson, E. J. Chem. Soc., Faraday Trans. 1998, 94, 1657. (20) Demetriades, K.; McClements, D. J. J. Agric. Food Chem. 1998, 46, 3936. (21) Chen, J.; Dickinson, E. J. Agric. Food Chem. 1998, 46, 91. (22) Mohan, S.; Narsimhan, G. J. Colloid Interface Sci. 1997, 192, 1. (23) Dalgleish, D. G. Trends Food Sci. Technol. 1997, 8, 1. (24) Dalgleish, D. G. Food Res. Int. 1996, 29, 541. (25) Magdassi, S.; Vinetsky, Y. Drugs Pharm. Sci. 1996, 73, 21. (26) Dalgleish, D. G. Bioprocess Technol. 1996, 23, 447. (27) Fang, Y.; Dalgleish, D. G. J. Colloid Interface Sci. 1993, 156, 329. (28) Dickinson, E. J. Chem. Soc., Faraday Trans. 1992, 88, 2973.
However, much less is known about the interactions of proteins with silicone-based polymers or surfactants. The unusual structural features of poly(dimethylsiloxane) molecules contribute to their impressive surface activity. Hydrophobicity is conferred by the methyl groups, and the bonding arrangement around the Si-O-Si linkage leads to extraordinary flexibility of the polymer backbone.31 This combination of properties allows silicones to easily migrate to and reside at interfaces, particularly air interfaces. The surface activity of silicones has been widely exploited via the development of silicone surfactants,32 and many studies have examined the behavior of silicones at solid surfaces and liquid and air interfaces.32-34 While silicone surface activity, and particularly hydrophobicity, are widely beneficial, this is not universally the case. There are many reports that poly(dimethylsiloxane)s, including small cyclic structures such as octamethylcyclotetrasiloxane (D4), and related silicones have the ability to denature proteins that come into contact with them.35-37 It is believed that in order for a protein molecule to favorably associate with oligo- or poly(dimethylsiloxane)s (PDMS), the protein must unfold such that the hydrophobic residues, which are generally sequestered in the center of the protein structure, are exposed to the external surface. This change in protein conformation permits the protein and silicone to interact via hydrophobic interactions at the cost of the protein’s tertiary structure. However, generally concomitant with the change in protein tertiary structure is a loss of (29) Dickinson, E. In Proteins in Solution and at Interfaces in Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993. (30) Ananthapadmanabhan, K. P. In Protein-Surfactant Interactions in Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993. (31) Brook, M. A. Silicon in Organic, Organometallic, and Polymer Chemistry; Wiley: New York, 2000; Chapter 9. (32) Schlachter, I.; Feldmann-Krane, G. Surfactant Sci. Ser. 1998, 74, 201. (33) Gasperlin, M.; Kristl, J.; Smid-Korbar, J.; Kerc, J. Int. J. Pharm. 1994, 107, 51. (34) Linden, M.; Rosenholm, J. B. Langmuir 2000, 16, 7331. (35) Darst, S. A.; Roberston, C. R.; Berzofsky, J. A. J. Colloid Interface Sci. 1986, 111, 466. (36) Anderson, A. B.; Roberston, C. R. Biophys. J. 1995, 68, 2091. (37) Sun, L.; Alexander, H.; Lattarulo, N. Biomaterials 1997, 18, 1593.
10.1021/la025867k CCC: $22.00 © 2002 American Chemical Society Published on Web 10/19/2002
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enzymatic activity. Even the adsorption of proteins to (hydrophilic) silica has been shown to alter the conformation of the protein.38,39 The interaction between silicones and proteins is not always detrimental to protein structure, however.40-46 Silicone-modified starch microparticles containing human serum albumin (HSA) have been generated using either a trimethylsilyl-terminated poly(dimethylsiloxane) 1 (PDMS) or a triethoxysilyl-functionalized poly(dimethylsiloxane) 2 (TES-PDMS, Chart 1). Only the proteins in the microparticles modified by the latter silicone were able to elicit an immune response after oral administration to mice. This suggests that the hydrophilically functionalized silicone was able to stabilize or protect the protein from degradation in vivo, whereas the unfunctionalized silicone could not. These and subsequent results40,42,43 established that a favorable physical or chemical interaction between a protein and a hydrophilically modified silicone is possible. The objective of the current research was to establish the rate and magnitude of protein deactivation on contact with normal silicone oil and the degree to which functional groups on a silicone surfactant mitigate this behavior. To facilitate a determination of these relationships, a simpler system involving only one (oil/water) interface was used rather than the complex microparticle system that has multiple liquid/solid, oil/water interfaces. The chosen commercial surfactant DC3225C (3) (Chart 1) possesses hydrophilic, but not chemically reactive, side chains. In addition, a system was used that would permit a clearer picture of the activity of the proteins. Thus, water-insilicone oil emulsions were used to examine the interaction of proteins with silicones: the enzymatic activities of proteins that had been emulsified were compared with enzyme controls that had not been exposed to silicone. Experimental Section Reagents. R-Chymotrypsin, benzoyl-L-tyrosine ethyl ester (BTEE), and lysozyme (Sigma, Inc.) were used without further purification. Dried Micrococcus lysodeikticus cells were supplied by Worthington Biochemicals. Octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and the surfactant DC3225C were obtained from Dow Corning and were used without further (38) Wan, R.; Sun, S.; Bekos, E. F.; Bright, F. V. Anal. Chem. 1995, 67, 149. (39) Welzel, P. B. Thermochim. Acta 2002, 382, 175. (40) Bartzoka, V.; Chan, G.; Brook, M. A. Langmuir 2000, 16, 4589. (41) Brook, M. A.; Jiang, J.; Heritage, P., Bartzoka, V.; Underdown, B.; McDermott, M. R. Langmuir 1997, 13, 6279. (42) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Langmuir 1998, 14, 1887. (43) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Langmuir 1998, 14, 1892. (44) Brook, M. A.; Zelisko, P. M.; Walsh, M. J.; Crowley, J. N. Silicon Chem., in press. (45) Zelisko, P. M.; Brook, M. A. Polym. Prep. (Am. Chem. Soc. Div. Polym. Chem.) 2001, 42 (2), 115. (46) Brook, M. A.; Zelisko, P. M. Polym. Prep. (Am. Chem. Soc. Div. Polym. Chem.) 2001, 42 (1), 97.
purification. HCl and tris(hydroxymethyl)aminomethane (Tris) used in buffer preparation were obtained from BDH, while K2HPO4, sodium bicarbonate, and absolute ethanol were supplied by Anachemia. The buffers comprising the aqueous phase of the emulsion were formulated using deionized, organic-free, distilled water containing 0.025% sodium azide (Aldrich). Calcium chloride, hydroxylamine, and Sephadex G-25 were obtained from Aldrich. Fluorescein 5-isothiocyanate (FITC) was purchased from Molecular Probes and used without further purification. Emulsion Mixing Vessel. All emulsions were formulated in a Pyrex 180 mL beaker model number 1140. The bottoms of the upper four mixing blades (pitched at a 45° angle) were positioned 3.3 cm from the bottom of the mixing vessel, and the bottom of the lower blades (at a 90° angle) were positioned 1.0 cm from the bottom of the mixing vessel. Emulsion Formulation for Enzyme Stability Studies. The silicone surfactant DC3225C (1.0 g) was dissolved in D4 (29.2 g) (continuous phase) at a concentration of 3.54 wt % and added to the mixing vessel. The R-chymotrypsin (final concentration 5.13 × 10-4 g/mL) was dissolved in 0.08 M Tris-HCl buffer (pH 7.8) which was also 1.0 M in CaCl2. Lysozyme was dissolved in 0.1 M potassium phosphate buffer (pH 7.0) to give a final concentration of 5.0 × 10-4 g/mL. These solutions constituted the dispersed phase of their respective emulsions. All enzyme solutions were prepared on the day of emulsification. Using a 30 gauge needle fixed to a 20 mL syringe, the aqueous dispersed phase (10.0 g), containing the desired enzyme, was added dropwise to the silicone continuous phase over a period of 10 min. The addition of the aqueous phase took place under dual blade, turbulent mixing conditions at 2780 rpm using a Caframo BDC6015 mixer. The emulsion was allowed to stir for an additional 20 min following addition of the dispersed phase. A portion (3.0 g) of the original enzyme stock solution was stored in a sealed container at room temperature as a control. Sizing of Emulsion Droplets. The diameter of the emulsion droplets was ascertained using a Nikon Labophot-2 optical microscope against polystyrene standards (Polyscience, Inc.), as well as a Coulter LS-230 particle sizer. Enzyme Extraction. The enzymes were extracted from the emulsion prior to the assessment of the enzyme activity. To extract the enzyme, a 5 mL aliquot of the emulsion was centrifuged at 2500 rpm (20 °C) for 60 min using a Beckman J2-21 centrifuge, and the supernatant silicone oil was discarded. The concentrated emulsion was transferred to the mixing vessel. Tris-HCl buffer (2.0 mL) was added dropwise to the concentrated emulsion at a rate of 0.1 mL/min while stirring at 3000 rpm. The mixture was then allowed to stir for an additional 30 min following the addition of the buffer. After extraction, the aqueous phase was drawn into a 5.0 mL syringe, filtered through a 0.2 µm syringe filter into a vial, and stoppered. Benzoyl-L-tyrosine Ethyl Ester (BTEE) Solution. The BTEE substrate solution was prepared by combining 6.3 mL of absolute methanol with 5.0 mL of deionized, organic free water. BTEE (3.79 × 10-3 g, 12.0 mmol) was added to this solution and dissolved. Fresh substrate solutions were prepared each day that an activity assay was performed. Activity Assay for r-Chymotrypsin. Enzyme activity assays were performed on days 1, 3, 5, 7, and 9 following emulsification using a Cary 400 spectrophotometer. Concentrations of enzyme extracted from the emulsion were calculated using Beer’s Law
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Figure 3. Enzymatic activity of R-chymotrypsin entrapped in a water-in-silicone oil emulsion containing DC3225C as a surfactant, as compared with the control R-chymotrypsin buffered aqueous solution over the same time period.
Figure 1. (A) Effect of dual-blade turbulent mixing stress on the activity of lysozyme in a buffered solution (no silicone present). (B) Enzyme activity following exposure of a buffered solution to D4 (no other materials present).
Figure 2. Photomicrograph of a water-in-silicone oil emulsion containing DC3225C as the surfactant. and an extinction coefficient () of 26 500 M-1 cm-1 at 256 nm.47 The concentrations of extracted enzymes were normalized with the control. Assays were performed by adding Tris-HCl (1.25 mL) to BTEE solution (1.25 mL) in a quartz absorbance cuvette and equilibrating at room temperature. Distilled, deionized water (10.0 µL) was added to the blank, and the enzyme solution (10.0 µL, extracted enzyme or control) was added to the sample cuvette. The increase in absorbance at 256 nm was measured for 10 min to determine enzyme activity (Figure 3). Activity Assay for Lysozyme. Dried M. lysodeikticus cells (9.0 mg) were dissolved in potassium phosphate buffer (30.0 mL, 0.1 M, pH 7.0). The cell suspension (1.990 mL) was pipetted into a polystyrene absorbance cuvette and equilibrated at room temperature for 5-10 min. To a cuvette containing the cell suspension was added enzyme solution (10.0 µL), and the decrease in absorbance of the Micrococcus cells was measured at 450 nm for 10 min. The slope of the initial linear portion of the plot was taken as the enzyme’s activity. Lysozyme concentrations were determined using Beer’s law and an extinction coefficient () of 38 000 M-1 cm-1 at 281 nm using UV/visible spectrophotometry.48 (47) The extinction coefficient was determined experimentally using Beer’s law and a known concentration of the protein. (48) Aune, K.; Tanford, C. Biochemistry 1969, 8, 4579.
Enzyme activity measurements were acquired on days 1, 3, 5, 7, and 9 following emulsification (Figure 4). As a control, the decrease in absorbance of a solution of M. lysodeikticus cells to which enzyme was not added was also measured. This natural flocculation rate was then compared with the M. lysodeikticus trials involving enzyme. Thermal Denaturation of r-Chymotrypsin and Lysozyme. Solutions (50.0 mL) of lysozyme (1.3 × 10-3 g/mL) in potassium phosphate buffer (0.1 M, pH 7.0) and R-chymotrypsin (5.0 × 10-4 g/mL) in Tris-HCl buffer (0.08 M, pH 7.8), respectively, were prepared. Each of these enzyme solutions (47.0 mL) was transferred to a round-bottomed flask and refluxed at 100 °C for 2.0 h to ensure complete denaturation of the protein, while the remaining 3.0 mL was stored in a sealed container at room temperature as a control. Appropriate enzyme assays (vide supra) were used to determine enzyme activity following the denaturation process. Mixing Stress on r-Chymotrypsin and Lysozyme. Separate solutions (50.0 mL) of R-chymotrypsin (5.0 × 10-4 g/mL) and lysozyme (1.3 × 10-3 g/mL) were formulated using TrisHCl buffer (0.08 M, pH 7.8) and potassium phosphate buffer (0.1 M, pH 7.0), respectively. Each enzyme solution (40.0 mL) was subjected in turn to the mixing conditions noted above; the remaining 10.0 mL was stored as a control. Note that the addition and mixing protocol followed that used for the emulsions but in the absence of silicone oil or silicone surfactant. The enzyme solutions respectively were allowed to stir for a total of 60 min at 2780 rpm and 60 min at 3000 rpm, under dual-blade, turbulent mixing conditions. To determine enzyme activity after the application of the mixing stress, the respective activity assays for the enzymes (vide supra) were performed (Figure 1A). Mixing of Aqueous Enzyme with Octamethylcyclotetrasiloxane. Solutions (15.0 mL) of R-chymotrypsin (final concentration 4.8 × 10-4 g/mL) and lysozyme (1.3 × 10-3 g/mL) were prepared in their respective buffers. These R-chymotrypsin or lysozyme solutions (10.0 g) were added dropwise, respectively, over a period of 10 min to D4 (30.0 g, 0.1 mol) in the mixing vessel. The remainder of the enzyme solution was stored in a sealed container as a control. Addition of the enzyme solution occurred under the same stirring conditions as outlined above for the emulsion formulation for enzyme stability studies. The mixture was allowed to stir for a total of 45 min. A separatory funnel was used to extract the lower aqueous layer from the silicone oil layer to facilitate recovery of the aqueous enzyme. The activity assays outlined above for R-chymotrypsin and lysozyme above were used to measure the activity of the enzyme subjected to the D4 versus that of the control (Figure 1B). Protein Labeling. A flame-dried 10 mL round-bottomed flask with stir bar was charged with R-chymotrypsin (0.0128 g), which was subsequently dissolved in 1.0 mL of sodium bicarbonate buffer (0.1 M, pH 8.5). FITC (100 mg, 0.257 mmol) was added to a flame-dried 25 mL round-bottomed flask and dissolved in 10 mL of anhydrous dimethylformamide (DMF) immediately before use. To the protein solution was added the FITC solution
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Figure 4. (A) Enzymatic activity of lysozyme entrapped in a water-in-silicone oil emulsion, containing DC3225C in the oil phase as a surfactant, compared with lysozyme in solution (control) over the same time period. (B) Plot of control data × 100. Table 1. Confocal Micrographs of Water-in-Silicone Oil Containing FITC-labeled Enzymes
(100 µL), and the solution was allowed to stir at ambient temperatures using a magnetic stirrer for 60 min. Denaturation of the protein was not observed. A 1.5 M solution of hydroxylamine was prepared by dissolving hydroxylamine (1.05 g, to give 210 mg/mL) in 5.0 mL of distilled, deionized water. Sodium hydroxide (5.0 M) was used to adjust the pH to pH 8.5. The volume of the hydroxylamine solution was subsequently doubled using distilled, deionized water, and the solution was incubated at room temperature for 60 min with stirring at which time the hydroxylamine solution (0.1 mL) was added to the reaction, which was allowed to stir for 10 min at room temperature to remove any unreacted FITC. The protein was then purified using a 10 × 300 mm column with Sephadex G-25 equilibrated in 0.1 M sodium bicarbonate buffer (pH 8.5) as the stationary phase using the sodium bicarbonate buffer as the mobile phase. The first fluorescent band to elute was the labeled protein. The labeled protein solution constituted the aqueous phase of several separate water-in-silicone oil emulsions that were formulated using the procedure outlined above. The mass of the aqueous phase was adjusted to 10.0 g with 0.1 M sodium bicarbonate buffer (pH 8.5), as necessary prior to emulsification, to give a solution containing 5.13 × 10-4 g/mL R-chymotrypsin. These emulsions were subsequently imaged using a Zeiss LSM 510 confocal microscope. The same procedure was used to react lysozyme (0.0240 g) dissolved in sodium bicarbonate buffer (20.0 mL) with FITC (100 mg) dissolved in DMF (10.0 mL). This resultant FITC-modified lysozyme was then purified (vide supra) and diluted to a final concentration of 1.2 × 10-4 mg/mL lysozyme and 5.0 × 10-6 mg/ mL FITC as judged by UV/visible spectrophotometry. Other solutions with different protein and labeled protein concentra-
tions were similarly prepared and subsequently imaged using confocal microscopy (Table 1).
Results and Discussion Two different enzymes, R-chymotrypsin and lysozyme, were explored in these experiments. These enzymes were selected for two reasons: (i) The enzymes are very different in terms of their size, hardness, and catalytic activity. (ii) Well-developed spectrophotometric activity assays are available for both of these enzymes.49-54 Thermal denaturation of the enzymes was performed as a means of determining how the respective enzyme assays would react to a denatured protein (negative control). A temperature of 100 °C was chosen to denature the enzymes, since this value is well above the temperatures at which lysozyme and R-chymotrypsin have been reportedly denatured.55-57 Heat-treated R-chymotrypsin (49) Berezin, I.; Martinek, K. FEBS Lett. 1970, 8, 261. (50) Baumann, W.; Bizzozero, S.; Dutler, H. FEBS Lett. 1970, 8, 257. (51) Hummel, B. Can. J. Biochem. Phys. 1959, 37, 1393. (52) Bentley, R. Methods Enzymol. 1966, 9, 86. (53) Pollock, J.; Sharon, N. Biochem. Biophys. Res. Commun. 1969, 34, 273. (54) Prager, E.; Wilson, A.; Arnheim, N. J. Biol. Chem. 1974, 249, 7295. (55) Brandts, J.; Lumry, R. J. Am. Chem. Soc. 1961, 83, 4290. (56) Hirai, M.; Arai, S.; Iwase, H.; Takizawa, T. J. Phys. Chem. 1998, 102, 1308.
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produced a slope of zero when subjected to the BTEE assay, and this was taken as an indication of the enzyme in a denatured state. Although a decrease in the absorbance of the Micrococcus assay was observed with the heattreated lysozyme, this decrease was attributed to the natural flocculation rate of the Micrococcus cells in a buffer solution rather than some portion of the enzyme in solution retaining its native conformation. The natural sedimentation rate of the Micrococcus cells was measured independently of the actual enzyme assay to verify this proposition. The mechanical energy involved in emulsification and extraction/demulsification of the protein can result in partial or complete denaturation of proteins, as commonly occurs when ovalbumin in egg whites is beaten to make meringue. Dual-blade turbulent agitation of an aqueous solution of R-chymotrypsin or lysozyme, in the absence of any silicone, was thus expected to result in the denaturation of the enzymes, which would be evidenced by a moderate to severe decrease in enzyme activity using the appropriate assay. Emulsions, of course, were not generated during the stirring of the aqueous enzyme solutions in the absence of silicone oil. As expected, the residual enzymatic activity of the R-chymotrypsin following the agitation process was insignificant. However, unlike the chymotrypsin, the lysozyme subjected to the mixing stress exhibited activity that was three times higher than that of the control (Figure 1A). Instances of dilute solutions of lysozyme exhibiting enhanced activity over controls have previously been reported in the literature.58 Thus, the effect of simple mixing on the enzyme activity was not predictable. When either aqueous R-chymotrypsin or lysozyme was mixed with D4 in the absence of functionalized poly(dimethylsiloxane) surfactant, significant deactivation of the enzyme was observed as expected (Figure 1B): stable emulsions were not formulated under these conditions. After coming into contact with D4 for a period of 45 min, the enzymatic activity of R-chymotrypsin decreased by 89% while the activity of the lysozyme decreased by 50%. Placing the enzyme in proximity to molecules of D4, in the absence of a surfactant, results in the transient adsorption of the enzyme at the silicone oil interface.37 Thermodynamics frequently favor rearrangement of the enzyme tertiary structure so that the internal hydrophobic domains of the enzyme can be exposed to the hydrophobic interface.59 The catalytic activity can be compromised when resulting changes in conformation affect the active site of the enzyme. These results correlate well with those reported in the literature for both liquid59,60 and solid interfaces.35-37 Two sets of experiments were undertaken to determine if the protein, which can be necessary for colloidal stability,44-46 resided preferentially at the water/oil interface in these emulsions. In the first, the protein concentration was held fixed at about 1 × 10-5 mg/mL and the amount of a fluorescent label (FITC) varied between 10-3 and 10-8 mg/mL: these solutions were then formulated into emulsions. In the other emulsions, labeled protein concentrations varied from 10-4 to 10-6 mg/mL; the label concentrations varied between 10-6 and 10-8 (57) Edwards, J. V.; Setumadhavan, K.; Ullah, A. H. J. Bioconjugate Chem. 2000, 11, 469. (58) Kaulins, U.; Mikelsone, V.; Anderson, P. P.; Valtere, A.; Vecmuktane, L. LLA Raksti 1991, 272, 23. (59) Hickel, A.; Radke, C. J.; Blanch, H. W. J. Mol. Catal., B: Enzym. 1998, 5, 349. (60) Ross, A. C.; Bell, G.; Halling, P. J. J. Mol. Catal., B: Enzym. 2000, 8, 183.
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mg/mL. Confocal microscopy was used to image these emulsions (Table 1). At higher FITC concentrations (>10-3-10-4 mg/mL), so much fluorescent light was emitted from the emulsion droplets that it was impossible to establish any features of the protein distribution in the droplet (Table 1A). By contrast, at FITC concentrations below 10-8 mg/mL, only diffuse light radiated back, providing little information about the structure of the emulsion droplets (Table 1B). At intermediate concentrations, preferential association of the protein with the W/O interface could be clearly seen by the fluorescent corona at the interface (Table 1C). This result suggests that the proteins within the emulsion droplets reside at the interface and interact with the surfactant to some extent, which provides the key to the (enhanced) stabilization of the proteins in the emulsion. Emulsions formulated with Dow Corning surfactant DC3225C were stable systems with emulsion droplets ranging in size from 1 to 10 µm in diameter with the mean diameter being between 2 and 5 µm (Figure 2) as compared with polystyrene standards using optical microscopy and a Coulter LS-230 particle sizer. Over time, the emulsion droplets settled to the bottom of the sample tube as a result of a density difference but were easily resuspended in the continuous phase prior to performing the demulsification procedure. Experiments were conducted in triplicate to ascertain the reproducibility of the observations. As a result of the opaque nature of the emulsion, it was not possible to accurately measure enzyme kinetics colorimetrically in situ. Therefore, the entrapped proteins were removed from the emulsion to assay the enzymes using UV/visible spectrophotometry. To do so, mechanical energy comparable to that used during emulsification was used with additional water to break the emulsion into two bulk phases. Once the water droplet structure had been removed, enzymatic activity could easily be ascertained using UV/visible spectrophotometry of the extracted aqueous phase. Enzyme activity is affected by a variety of factors in these systems. Even in buffer under relatively ideal conditions, the activity of enzymes degrades with age. This was observed to be a minor, but observable (Figure 4), effect in these water-in-silicone oil emulsion systems. By contrast, as has been previously observed, exposure of enzymes to silicone oil D4 catastrophically degrades enzyme activity. Mechanical energy, such as that used for emulsification and demulsification, also has a profound, frequently detrimental, effect on enzyme activity as seen with chymotrypsin (Figure 1). By contrast, lysozyme was found to be more active, presumably due to protein deaggregation, as has been previously observed.58 In summary, enzyme activity is generally slowly or rapidly eroded by external stimuli. The detrimental effects on enzymatic activity of aging, turbulent mixing, and exposure to silicone oil were completely muted when emulsification of protein/buffer was carried with silicone oil containing the siliconepolyether surfactant. The enzymatic activity of the emulsion-entrapped enzyme, after extraction, was no worse than that of the control for the case of R-chymotrypsin (Figure 3) and was significantly higher than that of the control in the case of lysozyme (Figure 4). This latter observation is consistent with the complementary effects of protection of the enzyme by the surfactant and activation of the enzyme due to mechanical agitation during emulsion (de)formulation.
Stabilization of Enzymes in Emulsions
Many organic functional groups61-67 have been demonstrated to retard protein adsorption at surfaces, with poly(ethylene oxide) (PEO)68-73 being one of the most effective. In the case of the PEO-silicone-stabilized emulsions, there are favorable interactions that hold the protein at the surfactant/water interface. These interactions must be quite significant: the mechanical energy used to make the emulsion, which was shown to dramatically reduce enzymatic activity of R-chymotrypsin in the absence of silicone, had no effect on the enzyme in contact with DC3225C. However, the protein/surfactant interactions are not sufficiently strong to result in significant changes in tertiary structure of the protein, at least as far as enzymatic activity is concerned. We and many others have demonstrated that protein/ nonfunctional silicone interactions are detrimental to protein stability. Thus, in the emulsions, the DC3225C surfactant must sequester the proteins such that they do (61) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841. (62) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927. (63) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336. (64) Boeckl, M. S.; Bramblett, A. L.; Hauch, K. D.; Sasaki, T.; Ratner, B. D.; Rogers, J. W., Jr. Langmuir 2000, 16, 5644. (65) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464. (66) Tidwell, C. K.; Ertel, S. I.; Ratner, B. D. Langmuir 1997, 13, 3404. (67) Deng, L.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 5136. (68) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (69) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J. Am. Chem. Soc. 2000, 122, 8303. (70) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J. Colloid Interface Sci. 1998, 202, 507. (71) Van Alstine, J. M.; Malmsten, M. Langmuir 1997, 13, 4044. (72) Malmsten, M.; Van Alstine, J. M. J. Colloid Interface Sci. 1996, 177, 502. (73) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. 1998, 102, 426.
Langmuir, Vol. 18, No. 23, 2002 8987
not come into contact to a significant degree with either the silicone backbone of the surfactant or with the silicone oil (D4) continuous phase. As a result, the enzymes are kept within a hydrophilic, locally viscous environment and denaturation by the hydrophobic silicone is inhibited. Thus, a marvelous balance exists at the water/oil interface. The enzymes adhere to the PEO at the interface, are stabilized against irreversible structural distortion by applied mechanical energy, and are protected from contact with the continuous silicone oil phase but are not otherwise affected, at least not in a way that moderates catalytic activity. Conclusions Despite the presence of denaturants such as silicones35-37 and mechanical stress, R-chymotrypsin and lysozyme entrapped within water-in-silicone oil emulsions display enzymatic activity that is equal to, or better than, that of the respective controls over the experimental time frame observed. The silicone surfactant acts as a physical buffer against the mechanical stress exerted on the biomolecules by the dual-blade, turbulent mixing system, as well as acting as a physical barrier against contact with the cyclic silicone present in the external phase of the emulsion. It may further act to stabilize the protein against denaturation due to favorable physical interactions at the oil/ water interface. Thus, these water-in-silicone oil emulsions can be used to effectively entrap, and possibly deliver, proteins or enzymes with comparable or less denaturation of the enzyme when compared to aqueous controls. Acknowledgment. Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes for Health Research (CIHR) is gratefully acknowledged. We also appreciate helpful discussions with Paul Berti (McMaster). LA025867K
