Water-in-Silicone Oil Emulsion Stabilizing Surfactants Formed From

Jul 16, 2008 - Both HSA and TES-PDMS were essential for the formation of stable ... neither the TES-PDMS nor the HSA could stabilize the emulsion inte...
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Biomacromolecules 2008, 9, 2153–2161

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Water-in-Silicone Oil Emulsion Stabilizing Surfactants Formed From Native Albumin and r,ω-Triethoxysilylpropyl-Polydimethylsiloxane Paul M. Zelisko,*,‡ Kulwinder K. Flora,§ John D. Brennan, and Michael A. Brook* McMaster University, Department of Chemistry 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 Received February 29, 2008; Revised Manuscript Received May 9, 2008

Contact with hydrophobic silicones frequently leads to protein denaturation. However, it is demonstrated that albumin in water-in-silicone oil emulsions retains its native structure in the presence of a functional, triethoxysilylterminated silicone polymer, TES-PDMS. Both HSA and TES-PDMS were essential for the formation of stable water-in-silicone oil emulsions: attempts to generate stable emulsions using independently either the protein or the functionalized silicone as a surfactant failed. Confocal microscopy indicated that the human serum albumin (HSA) preferentially adsorbed at the oil/water interface, even in the presence of another protein (glucose oxidase). A variety of experiments demonstrated that the hydrolysis of the Si-OEt groups on the functional silicone occurred only to a limited extent, consistent with the absence of a covalent linkage between the silicone and protein, or of cross-linked silicones at the interface. The fluorescence spectra of HSA extracted from the emulsions, front-faced fluorescence experiments on the HSA/silicone emulsion itself, and HSA/salicylate binding studies all demonstrated that the stability of the water/oil interface decreased as the protein began to unfold: unfolding of the protein in the emulsion was slower than in aqueous solution. The experimental evidence indicated that the interaction between HSA and TES-PDMS is not associated with either homomolecular (HSA/HSA; TES-PDMS/TES-PDMS) interactions or with covalent linkage between two the polymers. Rather, the data is consistent with the direct binding of unhydrolyzed Si(OEt)3 groups to native HSA. The nature of these interactions is discussed.

Introduction The increasing availability of proteins in quantity and their utility as catalysts (enzymes) and as drugs is driving the development of strategies for their targeted and controlled delivery. We recently demonstrated that it is possible to generate stable water-in-silicone oil emulsions containing both human serum albumin (HSA) in the dispersed phase and triethoxysilylethyl-modified polydimethylsiloxane (TES-PDMS) (Figure 1) in the continuous phase (octamethylcyclotetrasiloxane, D4).1,2 Both the protein and the silicone must be present for a stable emulsion (in excess of 8 months in some cases) to be prepared; neither the TES-PDMS nor the HSA could stabilize the emulsion interface independently.1 It was of interest to determine if the emulsion could be broken in a controlled manner for effective protein delivery and, at the same time, whether the released proteins would be in their native structure. Hydrophobic silicones, including both linear and cyclic polydimethylsiloxanes,3 are usually good denaturants for a variety of proteins.4,5 However, the interaction of proteins with hydrophobic materials including organic lipids (e.g., in foods6,7) and silicones need not necessarily be deleterious to the biomolecule: beneficial interactions between proteins and silicones have found numerous applications in the cosmetics and personal care industries.8 Several proteins are not affected by contact with certain hydrophilically modified silicone surfactants, * To whom correspondence may be addressed. Tel.: +1-905-6885550 ext. 4389. Fax: +1-905-682-9020. E-mail: [email protected] (P.M.Z.); [email protected] (M.A.B.). ‡ Current address: Brock University, Department of Chemistry, 500 Glenridge Avenue, St. Catharines, Ontario L2S 3A1. § Current address: HORIBA Jobin Yvon IBH Ltd., SkyPark 5, 45 Finnieston Street, Glasgow G3 8JU, United Kingdom, kulwinder.sagoo@ ibh.co.uk.

including polyalkylene oxide-modified silicone with a comblike structure (e.g., DC3225C, Figure 1).3,9 For example, lysozyme and R-chymotrypsin entrapped within water-insilicone oil emulsions made with this surfactant retained their ability to process substrate over a nine-day period at levels comparable to that of controls.3 In both of these cases, however, the proteins were simply entrapped within a stable emulsion droplet and did not play a significant role in generating a stable interface; Anseth et al. have demonstrated that DC3225C can effectively stabilize a water/silicone oil interface in the absence of any other additives.9 For the purposes of protein delivery, we judged these emulsions to be too stable. The silicone TES-PDMS is significantly different from the polyether DC3225C, possessing only a small, functional triethoxysilyl moiety at each terminus of a 28000 MW silicone chain;1 there are otherwise no polar groups. Human serum albumin (HSA) is a globular, surface-active protein that has been shown to adsorb at a number of different interfaces;10 it is not sufficiently surface active in its own right to stabilize a silicone/ water interface. Although a number of emulsion systems have been formulated using the combination of TES-PDMS and HSA at the oil/water interface, the role of the protein at this interface, if any, was unclear.1,2 A series of studies was designed to determine if HSA lies at the silicone interface and to establish the nature of any interaction between the surfactant and protein that allowed the formation of reasonably stable emulsions. In addition, the degree to which the protein was undergoing unfolding in the silicone emulsion was determined. In particular, it was of interest to establish if such emulsions could be broken in response to a stimulus to deliver their protein load. We report that the stabilization of water-in-silicone oil emulsions by albumin/TES-PDMS is the consequence of the direct interaction between the two polymers leading to a surfactant, as shown by

10.1021/bm800226z CCC: $40.75  2008 American Chemical Society Published on Web 07/16/2008

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Figure 1. Chemical structure of DC3225C and TES-PDMS.

microscopy, fluorescence spectroscopy, GC-MS, and NMR and that denaturation of HSA destabilizes the emulsion.

Experimental Section Reagents. Human serum albumin (HSA) 96-99% purity, bovine serum albumin (BSA), ovalbumin (OVA), blue dextran, and Sephadex G-25 were purchased from Sigma. Triethoxysilane, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), and octamethylcyclotetrasiloxane (D4) were obtained from Aldrich, while the HCl, NaOH, and trishydroxymethylaminomethane (Tris) used for buffer preparations were obtained from BDH and used without further purification. The buffer comprising the aqueous phase of the emulsion was formulated using deionized, organic-free, distilled water containing 0.025% sodium azide (Aldrich). Vinyl-terminated polydimethylsiloxane (1000 cSt and 2-3 cSt) and platinum divinyl-tetramethyldisiloxane complex (3-5% platinum concentration, Karstedt’s catalyst) in vinyl-terminated polydimethylsiloxane (Gelest), Texas Red C2 maleimide, 2-(4-pyridyl)-5((4-(2-dimethylaminoethylaminocarbamoyl)-methoxy)phenyl)oxazole (PDMPO; 1 mM in DMSO), and 5-(bromomethyl)fluorescein (5-BMF; Molecular Probes) were used without further purification. Spectra were acquired for NMR on a Bruker AV200 (at 200 MHz for protons); for UV/visible on a Cary 400 UV/visible spectrophotometer; for fluorimetry on a SLM-Amico 8100 fluorimeter; for time-resolved fluorescence on a PTI laserstrobe fluorimeter (Photon Technologies Incorporated, London, ON, Canada); for infrared on a Biorad FTS-40 Fouriertransform infrared spectrometer (FT-IR); and for confocal microscopy on a Zeiss LSM-510 confocal microscope. Purification of HSA. HSA (1.04 g) was dissolved in 60 mL of TrisHCl buffer (1.0 M, pH 7.8). Blue dextran (1.0 g) was dissolved in 60 mL of Tris-HCl buffer (1.0 M, pH 8) and passed through a Sephadex G-25 size-exclusion column equilibrated with the same buffer solution. The number of fractions required to collect all of the indicator was noted. The protein solution was then passed through the same column and a number of fractions equal to those required to elute the blue dextran were collected. The purified protein was then lyophilized and stored in a sealed container at 4 °C until needed. Locating Proteins within Emulsions Utilizing Fluorescent Probes. HSA was labeled with 5-BMF and Texas Red, respectively, and glucose oxidase was labeled with 5-BMF using standard procedures.11 The labeled proteins served to facilitate the analysis of the location of the protein within the emulsion using confocal microscopy. r,ω-(Triethoxysilylethyl)polydimethylsiloxane 500 MW and 28000 MW (TES-PDMS28000). R,ω-(Triethoxysilylethyl)polydimethylsiloxane, 500 MW and 28000 MW, was synthesized by hydrosilylation of vinyltriethoxysilane with H-terminated silicones using previously described protocols.12 TES-PDMS28000: 1H NMR (300 MHz, CDCl3) δ 0.07 (2270H, s), 0.57 (8H, s), 1.28 (18H, t, J ) 7.0 Hz), 3.82 (12H, q, J ) 7.0 Hz) ppm. 13C NMR (50 MHz, CDCl3) δ 1.19 (-OSi(CH3)-), 14.22 (-CH2CH2CH2-), 18.47 (PMDS-CH2CH2CH2SiOR3), 22.51 (-Si(OCH2CH3)3, 34.30 (PMDS-CH2CH2CH2SiOR3), 58.53 ((-Si(OCH2CH3)3) ppm. 29Si NMR (60 MHz, CDCl3) δ -45.2 (-Si(OEt)3), -21.9 (O-SiMe2-O), 7.0 (alkyl-Si-O) ppm. Fluorescence Studies of Extracted HSA. Steady-state fluorescence measurements using the Amico fluorimeter were collected using 10

µM of extracted HSA (following the appropriate dilution) in Tris-HCl buffer (1.0 M, pH 7.8). Fluorescence spectra were acquired by exciting the sample at 295 nm, and collecting the emission spectra from 305 to 500 nm in 1.0 nm increments to monitor the lone tryptophan residue of HSA. A 4 nm bandpass and 0.05 s integration time were employed. Emission spectra were collected for the sample, control, and a TrisHCl buffer blank. The emission spectra pertaining to the Tris-HCl blank were subtracted from both the control and sample emission spectra. Polarizers were set to 0° during excitation and 90° for collecting emission spectra in order to minimize scatter. Time-resolved fluorescence intensity decay data were acquired using the PTI laserstrobe as previously described.13,14 Samples were excited at 295 nm with emissions collected under magic angle polarization conditions and passed through a monochromator (4 nm band-pass) set at 335 nm. The intensity data were collected into 25 ps time windows, starting 2 ns before the laser pulse arrived (to establish a prepulse baseline) and covering a 40 ns range. The instrument response function was collected by measuring the Rayleigh scattering of the laser pulse from water and was used to deconvolute the instrument response profile from the experimentally determined decay trace. Appropriate baseline offset and time-shift parameters were obtained by allowing these to be floating parameters in the fit. Front-Faced Fluorescence. Front-faced fluorescence was employed to monitor fluorescence signals of HSA within the opaque emulsions to avoid artifacts due to scattering. In these experiments, the opaque emulsion was placed in a triangular quartz fluorescence cuvette. The fluorescence data were collected by exciting at 295 nm, and recording the emission spectrum from 305-450 nm. Scans were collected in 1 nm increments with 4 nm band passes.15,52 Large Volume Emulsion Formulation. HSA (0.9 g) was dissolved in 1.0 M Tris-HCl buffer (30.0 mL, pH 7.8) to give a final protein concentration of 0.03 g mL-1 (dispersed phase). A portion of the aqueous dispersed phase was stored in a sealed container at ambient temperature as a control. Triethoxysilylethyl-terminated PDMS (4.0 g) was dissolved in D4 (16.0 g, continuous phase) in the mixing vessel (Pyrex 180 mL beaker, model number 1140). The bottoms of the upper four mixing blades (pitched at a 45° angle) of diameter 2.5 mm were positioned 3.3 cm from the bottom of the mixing vessel and the bottom of the lower blades (at a 90° angle and diameter of 3.0 mm) were positioned 1.0 cm from the bottom of the mixing vessel.3 The aqueous dispersed phase (20.0 g) was added to the silicone oil phase in a continuous, dropwise manner (0.11 mL/min using a Harvard Apparatus PHD 2000 syringe pump) over a period of 2 h under dual blade, turbulent mixing conditions at 2780 rpm using a Caframo BDC6015 mixer. The emulsion was allowed to mix for an additional 2 h following the addition of the dispersed phase. The resulting emulsion was stored in a sealed container at ambient temperature. A portion of the emulsion was removed for use of in situ fluorescence measurements of HSA stability. Small Volume Emulsion Systems. All 5 mL emulsions were formulated using a Dremel tool16 using established procedures.2 Hydrolysis of TES-PDMS. The emulsion mixing vessel was charged with TES-PDMS500 or TES-PDMS28000 (4.2 g, 1.4 × 10-4 mol, 10 wt %), respectively, which was subsequently dissolved in D4 (16.0 g, 0.05 mol, 40 wt %). The mixing blade was positioned as previously described

Stabilization of Water-in-Silicone Emulsions (see below).3 Tris-HCl (20.0 mL, 0.05 M, pH 7.95) was added using the large volume emulsion protocol. The system was allowed to stir for an additional 3 h following the addition of the aqueous phase. The water and D4 were removed in vacuo, and the residue was analyzed using 29Si NMR and FT-IR. Gas Chromatography Analyses of TES-PDMS28000 Hydrolysis Products. An emulsion was formed using the large volume emulsion protocol: TES-PDMS28000 (4.1 g, 1.4 × 10-4 mol, 10 wt %), D4 (16.0 g, 0.05 mol, 40 wt %), and HSA (0.61 g, 8.97 × 10-6 mol) in TrisHCl (20 mL, 0.05 M, pH 7.95). After stirring for 3 h, an aliquot (5 mL) of the emulsion was centrifuged and the supernatant oil was discarded. The extracted aqueous phase was separated from the silicone oil and transferred to a sample vial capped with a septum. The sample was heated at 60 °C for 60 min in an oil bath. A 100 µL aliquot of the headspace in the vial was analyzed immediately after heating using gas chromatography (GC) and gas chromatography coupled with a mass spectrometry detector (GC-MS). For the GC experiments, a flame ionization detector (FID) was employed as the oven temperature was raised from 30 to 100 °C at a rate of 1°/min. The oven temperature was increased from 40 to 100 °C at a rate of 2°/min for the GC-MS experiments. In both cases, the detector and injector temperatures were set at 250 °C. Emulsion Formulated with 5-BMF-Labeled HSA. The large volume format was used with HSA (0.612 g), 5-BMF-HSA (1.0 mL, 1.99 × 10-4 mg/mL), 1.0 M Tris-HCl buffer (19.0 mL, pH 7.8), D4 (16.1 g), and TES-PDMS (4.14 g). When larger amounts of BMF label were present in these emulsions, clear confocal images could not be obtained. TES-PDMS/HSA Emulsions Containing PDMPO. The small volume format was used. A total of 3 mL of a solution of HSA (0.09 g, 1.32 × 10-6 mol) dissolved in 0.5 M Tris-HCl buffer (3.5 mL, pH 7.8), 1.0 mM PDMPO in DMSO (250 µL), was added over 5 min to TES-PDMS28000 (0.6 g, 2.14 × 10-5 mol) in D4 (2.0 g, 0.009 mol). The emulsion was allowed to mix for an additional 5 min following the addition of the aqueous phase. The remaining aqueous phase (0.5 mL) was kept as a control. Both the emulsion and the control were analyzed using front-faced fluorescence. PDMPO was incorporated into an emulsion containing DC3225C and examined in a pH 7.8 buffered solution as a control. Emulsion Formulated with Thermally Denatured HSA. HSA (0.59 g, 8.67 × 10-6 mol) was heated at 60 °C for 60 min and then emulsified (large emulsion format): TES-PDMS28000 (4.15 g, ∼1.4 × 10-4 mol), 1.0 M Tris-HCl buffer at pH 7.8 (20 mL), D4 (16.06 g, 54.3 mmol). Emulsion Formulated with HSA Denatured by Guanidine Hydrochloride. Crystalline HSA (0.912 g, 1.34 × 10-5 mol) was added to a guanidine hydrochloride solution (30.0 mL of a 10 M solution in 1.0 M Tris-HCl buffer, pH 7.8). The solution was allowed to stir at room temperature for a period of 15 min. Emulsification, using the large format, utilized denatured HSA solution (20.0 mL); TESPDMS28000 (4.016 g, 1.43 × 10-4 mol); D4 (16.017 g, 0.054 mol). Emulsion Formulated with HSA and a Saturated Solution of NaCl. HSA (0.604 g, 8.88 × 10-6 mol) was dissolved in a saturated sodium chloride solution (20 mL) and formulated into an emulsion (large volume format) using TES-PDMS28000 (4.063 g, 1.45 × 10-4 mol); D4 (15.998 g, 0.054 mol). The system was allowed to stir for an additional 3 h following the addition of the aqueous phase. Emulsion Formulated with HSA in a Solution of 8 M Urea as the Aqueous Phase. HSA (0.614 g, 9.03 × 10-6 mol) was dissolved in a solution of 8 M urea (20 mL) and formulated into an emulsion (large volume format) using TES-PDMS28000 (4.395 g, 1.57 × 10-4 mol); D4 (16.032 g, 0.054 mol). The emulsion was allowed to stir for an additional 3 h following the addition of the aqueous phase and examined by fluorimetry. Thermal Degradation of an HSA/TES-PDMS28000 Emulsion. HSA (0.61 g, 9.24 × 10-6 mol) was dissolved in 0.1 M Tris-HCl buffer (20 mL, pH 7.95) and formulated into an emulsion (large volume format)).

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TES-PDMS28000 (4.07 g, 1.45 × 10-4 mol); D4 (16.1 g, 0.054 mol). The emulsion was allowed to mix for an additional 3 h following the addition of the aqueous phase. Aliquots of the emulsion were heated at 60 °C for 6 and 24 h, respectively. A sample of the emulsion was kept in a sealed container at room temperature as a control. The HSA in the aqueous phase of each aliquot was examined for its extent of denaturation using front-faced fluorescence spectroscopy. Emulsion Formulated with Mixtures of Labeled HSA (Texas Red) and Glucose Oxidase (Fluorescein). Two labeled protein solutions (HSA (see above) 10 mL, glucose oxidase (see above) 10 mL) were combined and added to TES-PDMS28000 (4.1 g, 1.46 × 10-4); D4 (16.0 g, 0.054 mol). Following emulsification, the relative location of the two proteins in the emulsion was examined using confocal microscopy. Care was exercised to limit exposure to light during the entire course of the experiment. Protein Extraction for Assessment of HSA Stability. HSA was extracted from each emulsion to permit assessment of denaturation. To extract the protein, 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, pH 7.8) 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. Following the 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. The concentration of the protein in the aqueous solution was determined by spectrophotometry ( of HSA at 277 nm was taken as 36000 M-1 cm-1).17 Salicylate Binding to HSA. To a 10.0 µM solution of HSA (extracted protein or fresh HSA control) in Tris-HCl buffer (1.0 M, pH 7.8) in a poly(methyl methacrylate) fluorescence cuvette was added salicylate (10.0 µM in Tris-HCl buffer) in 10.0 µL increments. These same additions were made to a Tris-HCl blank. Following each addition of salicylate, the sample solutions and blank were allowed to stir for 1.0 min. Fluorescence spectra (305-500 nm) were acquired following each addition, and the blank spectra were subtracted from the spectra of the two protein containing solutions. The quenching of the lone tryptophan residue in HSA by the salicylate was used to monitor the binding efficiency of HSA.18

Results and Discussion Emulsion Preparation. Aqueous protein in silicone oil emulsions were prepared in two different formats, “large” (20-100 mL)1,19 or “small” (Dremel tool,16 2-5 mL), respectively:1 analogous processes should permit scaling from a few milliliters to several thousand liters. Emulsion droplets of 2-5 µm in diameter formed in either case and were stable for up to several months.1,2,19 The stability of these emulsions depended greatly on the protein utilized. It did not prove possible to form stable emulsions using lipase, lysozyme, glucose oxidase, or R-chymotrypsin, respectively, with TES-PDMS as surfactant (data not shown). These compounds represent several structural classes of protein. Stable emulsions, however, were readily formed from freshly prepared solutions of HSA, BSA, and OVA (Figure 2), although the emulsions were less stable than those prepared with silicone/polyether copolymer surfactants.3,19 Given the homology between the three albumin forms and the available benchmark data for the structural stability of HSA, HSA became the focus of the subsequent research. A series of experiments was undertaken to establish, where the HSA was located in the emulsion; the extent to which HSA undergoes unfolding during and after emulsification, and the types of interactions that are occurring between the protein, specifically HSA, and alkoxysilane-silicone surfactant (TES-PDMS).

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Figure 2. Optical micrographs of a TES-PDMS water-in-silicone oil emulsion containing BSA (left) and OVA (right) in the aqueous phase. The scale bar represents 20 µm.

Interfacial Characterization. The location of protein in the emulsions was established by confocal microscopy with HSA labeled with a fluorescent tag, 5-BMF. When unlabeled HSA/ buffer solution into which was doped a small amount of 5-BMFHSA (5.3 v/v%) was used, a corona of fluorescence was clearly observed in images of the central plane(s) of the emulsion droplets (Figure 3A). These data are consistent with HSA being located primarily at the oil/water interface rather than homogeneously distributed throughout the aqueous drop. The level of affinity of HSA (labeled with Texas Red C2 maleimide) for the interface was tested in the presence of a second protein; glucose oxidase labeled with 5-BMF. Glucose oxidase was chosen as the second protein in this system because it is not a proteolytic enzyme, ensuring that the HSA would not be cleaved during the course of the experiment20,21 A midline view of the droplet, using confocal microscopy, clearly shows that HSA preferentially adsorbed at the oil/water interface (Figure 3B, other droplets are more indicative of the outer surfaces of the emulsion droplets). This result, which is consistent with the well-known surface activity of HSA,22 suggests that HSA has a protective/isolating role at liquid interfaces as has been observed at solid surfaces.23,24 A variety of complementary interactions between HSA and TES-PDMS could lead to a stabilized water/silicone interface (Figure 4). The alkoxysilane is able to undergo nucleophilic substitution reactions by functional groups on the protein, water, or other silanols (leading to cross-linking), covalent interactions between the molecules must be considered. In addition, both molecules contain polar functional groups that are, or can be rendered, capable of hydrogen bonding; both have hydrophobic constituents and both molecules are able to undergo homo- (e.g., protein aggregation) or heteromolecular interactions. These possibilities were tested, first by tracking alkoxysilane hydrolysis and condensation. Trialkoxysilanes are routinely used to modify polar metal oxide surfaces,25 and as cross-linkers in room temperature vulcanization (RTV) silicone elastomer preparations.26 Thus, mechanisms of interfacial stabilization could include: (i) formation of protein/silicone complexes by nucleophilic displacement of Si-OEt groups by the protein, (ii) cross-linking of silicones at the interface to provide a protective elastomer shell, or (iii) protein interaction with hydrolyzed SiOH groups. Nucleophilic Protein Silicone Linkages. The protein contains a number of nucleophiles in the form of primary amines, alcohols, and thiol groups that in principle could react with the alkoxysilyl-modified silicone (protein-Nu + (EtO)3Si-silicone f protein-Nu-Si(EtO)2-silicone + EtOH). However, the resulting bonds: silazanes (Si-N, e.g., derived from lysine), silylsulfides (Si-S, e.g., derived from cysteine), and alkoxysilanes

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(e.g., derived from serine) are all equally, or less, stable to hydrolysis than the starting material.26 This suggests that such direct covalent interactions will only temporarily be viable in an aqueous environment, and that the silicone and protein interact primarily via physical interactions, rather than covalent bonds, to stabilize the interface of water-in-silicone oil emulsions. In addition, as noted below, such reactions would generate significant quantities of EtOH, which were not observed. Silicone Skin. The room temperature hydrolysis and condensation of alkoxysilanes (RTV) is most frequently catalyzed by tin or titanium esters,27 but may also be catalyzed by acid, base, and proteins.28,29 When the stability of HSA/TES-PDMS water/oil emulsions was tested by addition of an RTV catalyst, dibutyltin dilaurate, the initially stable emulsion immediately collapsed with the accompanying formation of visible, insoluble silicone elastomer “clumps” (data not shown). The immediate flocculation of the emulsion upon addition of the catalyst, leading to cross-linking, is inconsistent with a significant degree of pre-existing cross-linking at the interface of a stable emulsion.1 As noted below, 29Si NMR studies are also inconsistent with significant silicone cross-linking at the emulsion interface. Hydrolysis of AlkoxysilanessProtein-Silanol Interactions. Silanols are more acidic than alcohols, and it is conceivable that efficient hydrogen bonding interactions between proteins and silanol-terminated silicones could stabilize the water/oil interface; this would first require hydrolysis of the alkoxysilane to silanol (Figure 4D). A series of experiments were undertaken to test for alkoxysilane hydrolysis in the emulsions including probing for silanols using fluorescence or IR; examining 29Si NMR for the presence of silanols or disiloxanes; and, establishing the presence of ethanol in the head space above the emulsion by GC-MS. 2-(4-Pyridyl)-5-((4-(2-dimethylaminoethylamino-carbamoyl)methoxy)phenyl)oxazole (PDMPO)30 can be used to monitor the hydrolysis of alkoxysilanes31 to the corresponding silanol, by following a blue shift in the PDMPO fluorescence spectrum. Front-faced fluorescence of a white, macroscopically homogeneous, opaque HSA/TES-PDMS cosurfactant emulsion containing PDMPO (excited at 380 nm, emission spectrum was monitored from 350-650 nm) showed a small blue shift (Figure 5), suggesting that only partial hydrolysis of alkoxysilanes had taken place.31 The conclusion is further supported by IR and NMR data. Over time, a new absorption appeared in the region of 3300 cm-1 in the IR spectrum, which can be ascribed primarily to entrained water because the additional absorptions associated with silanol absorptions at 3580 cm-1 were not observed (see Supporting Information).32 The peak for Si1 in the 29Si NMR, whichshouldnotbeaffectedbyhydrolysisatSi2 ((EtO)3Si1(CH2)3Si2silicone), was used as an internal standard to gauge reaction. The ratio of the intensity of these peaks was found to be 1.880 in TES-PDMS28000 that had been freshly synthesized, and had not been exposed to buffer or to protein. After exposing the TES-PDMS28000 to 0.05 M Tris-HCl buffer (pH 7.95) for 6 h this ratio changed to 1.169, and the ratio further decreased to 1.123 following the exposure of the TES-PDMS28000 to a solution of HSA in 0.05 M Tris-HCl (pH 7.95). The decrease in this ratio indicates that the number of -Si(OEt)3 groups present in the system is changing, presumably as a result of hydrolysis. However, hydrolysis of the alkoxysilyl groups of the TESPDMS should produce an additional peak in the T-region (RSi(OR′)3) of the 29Si NMR spectrum, for the resulting silanol, located between -40 ppm and -80 ppm: 33 no new T type peaks were observed during these studies.

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Figure 3. (A) Water-in-silicone oil emulsion containing 5-BMF-HSA in addition to nonlabeled HSA with the arrows denoting the corona of protein at the oil/water interface. (B) Confocal microscopy images of a TES-PDMS water-in-silicone oil emulsion containing HSA (red) and glucose oxidase (green). The droplet in the lower right-hand corner represents a view at a midline focal plane of the emulsion droplet while the outer, surface focal plane of central droplet is depicted.

Figure 5. Fluorescence spectrum of PDMPO in 0.5 M Tris-HCl buffer (pH 7.8; ]) and in the aqueous phase of a TES-PDMS/HSA emulsion (b).

Figure 4. Possible TES-PDMS/HSA interactions at the silicone oil/ water interface.

Several attempts were made to follow these processes with more sensitive techniques that included hydrolysis in chloroform, using Cr(acac)3 as a relaxation agent for 29Si NMR: use of INEPT and HMBC techniques (see Supporting Information) and use of low molecular weight TES-PDMS500 to increase the relative proportion of end groups. In none of these cases, after exposure to either Tris-HCl or Tris-HCl + HSA, was it possible to observe either changes in signal ratios, correlation patterns, or the presence of new peaks (see Supporting Information). These data are consistent with only low levels of hydrolysis occurring (2 M NaClb heat: 60 °C for 6 h49,50 urea, 8 M (no TES-PDMS) urea, 8 M

5-15 min 3h 2 days 6h infinite 3 days

folded state from fluorescence data initially folded for the first 21 days, then gradual unfolding denatured denatured partially unfolded denatured denatured denatured partially unfolded

fluorescence emission maximumb (nm) 335 (@ t ) 0) 343 344 331 331 340 339 331

a TES-PDMS is present in all systems, unless otherwise stated. b Brine serves not as a denaturant, but to test the stability of the emulsion at high ionic strength conditions.

interaction between the protein and silicone polymer is responsible for stabilization of the silicone oil/water interface. Protein aggregation can stabilize water/air interfaces. For example, partially denatured ovalbumin protein forms a network with neighboring denatured protein molecules in a meringue.34 However, as noted below, fluorescent measurements showed essentially no denaturation of HSA in the initially formed, stable emulsion. Normally, aggregates of HSA, which were not observed, are found when the protein has been denatured to some extent.35 Thus, a different mechanism is operating. Protein:silicone Interactions: A Binding Domain on HSA for (EtO)3Si Groups?. Both Bassindale28 and Morse31 have explored the ability of a series of enzymes to catalyze the hydrolysis and condensation of Si(OEt)4 and MeSi(OEt)3. While not particularly effective, bovine serum albumin was demonstrated to be able to catalyze these processes, suggesting that BSA has a binding pocket for these alkoxysilane structures from which hydrolysis takes place.36–38 Based on the similar behavior between ovalbumin, BSA and HSA in silicone/water emulsions, the absence of evidence of a covalent linkage between TESPDMS and the protein, or of protein aggregates at the interface (vide supra), we speculate that HSA may bind directly to TESPDMS to generate a protein/silicone surfactant that can stabilize the water-silicone oil interface. HSA is an effective carrier protein that facilitates transport of a wide variety of materials in the blood: the binding of salicylate is just one example of its generic ability to bind molecules when in its native form.18,39 If binding occurs between

HSA and the Si(OEt)3, this may suggest the presence of a binding pocket for triethoxysilyl groups. In the case of small molecules such as Si(OEt)4 and MeSi(OEt)3, binding is followed by hydrolysis and condensation processes.40–42 If such binding is taking place between HSA and TES-PDMS, substantial hydrolysis does not result at the emulsion interface. The differences in reactivity with HSA between TES-PDMS and small alkoxysilanes such as Si(OEt)4 and MeSi(OEt)3 (hydrolysis and condensation) may be attributed in the former case to the highly hydrophobic silicone tail, which could retard hydrolysis through both steric and hydrophobic effects. The ability of the silicone, following binding to HSA, to effect reaction chemistry should be dependent on the local conformation of the protein, and should be readily manipulated by denaturing the protein. To test this proposal, we examined the effect protein unfolding on emulsion stability. Stability of the Interface: Relationship between HSA/ TES-PDMS Binding, Protein Folding, and Emulsion Stability. Silicones have been widely reported to facilitate the unfolding of proteins.3–5 The ability of the HSA to resist denaturation in the presence of TES-PDMS would be a guide to the efficiency of binding between the two polymers, and thus to the degree of stabilization of the emulsion interface. A comparison was therefore made between the stability of emulsions formulated with native protein and HSA that had been denatured either thermally or chemically (Figure 6, Table 1). Formulation of an emulsion with HSA containing 8 M urea, a concentration that is well-known to completely denature

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Figure 7. (A) Emission maxima of HSA extracted from the water-in-silicone oil emulsion ([) and HSA dissolved in a Tris buffer solution (9). (B) The extent to which salicylic acid quenches the tryptophan of emulsion-extracted HSA ([) and HSA in a buffered solution (9).

HSA,43,50 led to an emulsion that was stable for over three months. When the emulsion was subjected to analysis using front-faced fluorescence spectroscopy, the spectra revealed that the HSA had a λmax at 339 nm, consistent with partial denaturation (the λmax of denatured HSA is 345 nm, see Supporting Information). The emulsions were optically translucent as compared to the white, opaque emulsions formulated with HSA dissolved in Tris-HCl buffer. The stability of this emulsion, despite the evidence of denatured HSA, is perhaps not surprising given the ability of urea to behave as a chaotropic agent. This particular characteristic of urea would serve to stabilize the emulsion droplets against collisions and thereby retard Ostwald ripening of the emulsion.44–46 This type of phenomenon, using polymers rather than urea, was exploited by Marie et al. in their synthesis of polyaniline particles utilizing inverse emulsions.47 An emulsion formulated using HSA that had been denatured by 10 M guanidine hydrochloride began to demonstrate signs of phase separation within 3 h of formulation, a considerably shorter period of time than emulsions formulated with native HSA (minimum 1-3 months).1,2,19,48 Emulsions formulated using HSA dissolved in a saturated sodium chloride solution (>10 M) as the aqueous phase were initially white, opaque, and homogeneous in nature but formed two discrete phases after approximately 2 days. It is difficult in this case to separate out the relative contributions to emulsion instability of protein unfolding and salting out of the double layer. An emulsion prepared with thermally denatured HSA (60 °C for 1 h)49 separated into two phases shortly after emulsification (∼3 days). When a preformed, freshly prepared HSA/TES-PDMS emulsion was heated to 60 °C, phase separation was evident after 6 h, with complete phase separation occurring after 24 h. Fluorescence spectroscopy of the aqueous phases from these emulsions revealed that the HSA had denatured (Figure 6). These results suggest that the HSA, in the absence of any chaotropic additives in the aqueous phase of the emulsion, must retain its native conformation in order to optimally interact with the TES-PDMS and thereby stabilize the water/silicone oil interface in the emulsion system. This proposal was tested by following the kinetics of unfolding and emulsion destabilization. HSA in its native state is known to have an emission maximum (λmax) at 335 nm.50 As the protein begins to denature, owing to a slight expansion of domains I and II, a shift in the λmax to shorter wavelengths is observed, followed by a shift to

longer wavelengths (∼345 nm) as the tertiary structure of domain II in HSA unfolds.51,52 For the majority of the experiments, because the emulsions are opaque, it was first necessary to break the emulsion through the application of shear stress and addition of excess buffer. Once extracted from the emulsion, the fluorescence spectrum of the aqueous albumin phase was obtained by exciting the lone tryptophan residue of the protein (position 214 in domain II) at 295 nm and collecting the fluorescence data from 305 to 500 nm. Aliquots of aqueous protein were extracted from the emulsion over a 25 day period and the λmax was recorded (Figure 7A). The emission maximum of the HSA extracted from the emulsion did not differ significantly from that of the control (HSA dissolved in a Tris-HCl buffer solution) for the first 25 days of the trial. After this point in time, a noticeable blueshift could be observed in the protein’s emission spectrum, consistent with the formation of the expanded form of the protein. This shift in the spectrum coincided with the appearance of the first signs of phase separation in the emulsion (increases in average droplet size). Front-faced fluorescence experiments, using a triangular fluorescence cuvette, were performed directly on the emulsion to examine HSA stability when residing at the emulsion interface. The results were correlated with the behavior of emulsion extracted proteins. As shown in Figure 6, in the emulsion, HSA shows are slight blue shift with an emission spectrum essentially overlapping that of HSA which was heated for 6 h, suggesting that the more hydrophobic environment may lead to a slight expansion of the protein structure, or may directly affect the solvation of the Trp residue. To further probe the properties of HSA, the changes in emission wavelength of extracted HSA were correlated to the efficiency of HSA to bind salicylic acid. Binding of salicylic acid to native HSA leads to the quenching of the protein’s tryptophan emission at 335 nm, while the emission of salicylic acid becomes apparent at 408 nm.51 Salicylic acid can therefore be titrated into a solution of HSA and the extent to which the protein binds the salicylic acid can be monitored by recording the slope of the change in the tryptophan emission intensity. The change in Trp emission intensity upon addition of salicylic acid to the emulsion-extracted HSA was compared to that of HSA in a buffer solution, showing that both samples lost salicylic acid binding ability at the same rate, becoming unable to bind the ligand after 2 weeks. Loss of ligand binding ability is known to correlate with the formation of the expanded state

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Figure 8. (A) Mean fluorescence lifetimes of the emulsion-extracted HSA (9) and HSA in a buffered aqueous solution ([). (B) The quantum yield of the HSA extracted from the emulsion (9) and HSA in a buffered solution ([).

of HSA,13 and thus the changes in ligand binding and emission wavelength are relatively well correlated To further probe the conformational changes in the protein we examined both the emission lifetime and quantum yield of the protein after extraction from the emulsion and compared these values to those obtained from fresh HSA in aqueous solution. Both extracted and fresh HSA showed decreases in lifetime (Figure 8A) and quantum yield (Figure 8B) over a period of 2 weeks, which were in reasonable agreement with the loss of salicylate binding activity over this period. Thus it can be concluded that HSA undergoes the first step in the unfolding pathway (formation of an expanded state) over a period of about 2 weeks, during which time it loses ligand binding ability. The changes in the conformation of HSA correlate with a slowly degrading stability of the emulsion interface as demonstrated by increased droplet size and, eventually, complete collapse of the interface and macroscopic phase separation. However, the strong correlation between the unfolding of extracted and fresh HSA show that this unfolding is clearly not occurring as a consequence of direct contact between the HSA and silicone oil during formation, or of cracking of the emulsion, or at rest in a stable emulsion. Instead, the HSA is as stable or more stable at the interface than in the control sample. Triggered Emulsion Instability. The macrosurfactant created by the interaction between TES-PDMS and HSA stabilizes the silicone/water interface, although neither compound can perform this function alone. These data demonstrate that the albumin tertiary structure is related to emulsion stability and, by inference, the stability of the protein/silicone interface. However, these data provide neither rationalization for the protective nature of the interface against the normally denaturing interaction of HSA with silicone oil, nor the nature of silicone protein interactions at the interface that leads to stabilization of the emulsion interface. The experiments undertaken are not consistent with either cross-linking of the silicone or the interaction of partly hydrolyzed alkoxysilane (silanol) with HSA. It is possible, given the ability of HSA to act as a generic carrier in blood, that the triethoxysilyl groups are able to bind to pockets on the HSA. As the HSA denatures, the effectiveness of such binding would be reduced, leading simultaneously to a decrease in stability of the cosurfactant and of the emulsion interface. There is thus a synergistic interaction between the protein and TES-PDMS that results in emulsion stability. On formation,

a strong binding interaction at the emulsion interface occurs between HSA and TES-PDMS which stabilizes both the protein conformation and the emulsion interface (Figure 4E). As HSA unfolding begins to occur, the strength of the interaction with TES-PDMS begins to degrade, further accelerating both HSA unfolding and the destabilization of the emulsion. Irrespective of the mode of interaction, the HSA/TES-PDMS interaction must be highly effective because it limits HSA-silicone oil interactions, hindering HSA unfolding, even when the protein is exposed to very high levels of mechanical energy or heat. The key observations of this work are the ability to stabilize multiple proteins in emulsions without significant denaturation of the protein resulting from exposure to silicone. The higher affinity of HSA for the interface and sensitivity of the emulsion stability to denaturation of the HSA, unlike water-in-silicone oils stabilized with polyether silicone surfactants, provide a strategy to deliver proteins. It should be possible to entrap more than one protein in a water-in-silicone oil emulsion, use the preferential adsorption of HSA at the oil/water interface to protect the other proteins that reside in the bulk water of the emulsion droplet from exposure to silicone oil, and yet liberate all proteins by selectively targeting the denaturation of the HSA to break the emulsion. Investigations are ongoing to investigate this proposal.

Conclusions HSA and the triethoxysilyl groups on TES-PDMS interact to form a type of cosurfactant at silicone oil/water interfaces: both constituents are necessary to create a stable interface. The stability of this interface is lower than with good silicone surfactants, such as those based on alkylene oxide polymers. The interfacial stability is associated with the degree to which HSA undergoes unfolding. As unfolding occurs, as shown by a variety of fluorescent techniques, there is a concomitant loss of emulsion stability. The TES-PDMS/HSA interaction is remarkably protective against protein denaturation, both of HSA and other proteins present in the aqueous phase. In the absence of TES-PDMS, contact between HSA and silicone oil leads to complete denaturation of the proteins within hours: with the silicone surfactant, unfolding is retarded to the extent of weeks or months and the interaction is also protective against the significant mechanical energy needed to prepare and break the emulsions. The exact nature of the interaction between the silicone and the HSA molecules remain unclear, although

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covalent interactions, silicone cross-linking and protein aggregation have been discounted. The observations are consistent with a specific mode of binding between HSA and the Si(OEt)3 groups on the TES-PDMS polymer. Acknowledgment. Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) is gratefully acknowledged. We would also like to thank Dr. Brian McCarry and Cam Harrington of McMaster University for helpful discussions. J.D.B. holds the Canada Research Chair on Bioanalytical Chemistry. Supporting Information Available. Experimental details and supporting analytical data. This material is available free of charge via the Internet at http://pubs.acs.org.

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