Proteins at Silicone Interfaces - American Chemical Society

Chapter 16

Proteins at Silicone Interfaces 1,2

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Paul M . Zelisko , Amro M . Ragheb , Michael Hrynyk , and Michael A. Brook

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: August 2, 2007 | doi: 10.1021/bk-2007-0964.ch016

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Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada Current address: Department of Chemistry, Brock University, 500 Glenridge Avenue, Saint Catharines, Ontario L2S 3A1, Canada *Corresponding author: [email protected] 2

Although enzymes are normally exploited in aqueous, biological environments, certain enzymes can be made to operate in organic media. We report the utilization of enzymes in active form in a yet more hydrophobic silicone medium, in both liquid and elastomeric forms, by using suitable non-ionic surfactants. The enzymatic activity, depending on the protein, can be shown to be even higher than in organic media, if the concentration of water is appropriately controlled. α-Chymotrypsin, an enzyme readily denatured by simple silicone oils when bulk water is present, can be rendered highly active in both silicone oils and silicone elastomers when stabilized with poly(ethylene oxide) based surfactants: human serum albumin was similarly unaffected by the liquid silicone when surfactants were present. Lipase was shown to have a higher activity in silicone elastomers than in hydrocarbon solvents, but only if PEO constituents were not added. The factors necessary to stabilize protein structure and retain enzymatic activity, such as water content and the presence of PEO, in a silicone-rich environment are described.

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© 2007 American Chemical Society

In Science and Technology of Silicones and Silicone-Modified Materials; Clarson, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Introduction Siloxane polymers are among the most hydrophobic polymers available: only fluoropolymers have lower surface energies. Proteins in contact with exceptionally hydrophobic silicones can undergo unfolding, with consequent loss of activity in the case of enzymes. ' Two factors can mitigate these denaturing events: control of available water, and the presence of surface active molecules. Klibanov, Hailing, and others have shown that some proteins can operate effectively in organic solvents that have minimum water content. " At low water levels, internal hydrogen bonding interactions maintain protein tertiary structure, yet permit sufficient flexibility around the active site for reactions to be catalyzed. " An alternative approach in organic solvents stabilizes enzymes by using surfactants that control the degree to which the hydrophobe can come into contact with the protein. Thus, the addition of high molecular weight (>25000 MW) silicones, with a few hydrophilic groups, to the silicone oil/aqueous protein mixture can prevent protein denaturation, even though the protein is in close proximity to the silicone oil in an emulsion. We wished to develop silicone based emulsion and elastomeric systems in which proteins were stabilized for use as immobilized enzymes, or could be released on demand as bioactives. It was reasoned that control of both water content and the interfacial structure could be achieved using poly(ethylene oxide)(PEO) as a constituent. PEO is widely understood, to be a "proteinfriendly" polymer, as demonstrated by improved potency of protein drugs when "pegylated" and improved biocompatibility of surfaces when modified with PEO. ' Enzyme activities of two very different enzymes, ct-chymotrypsin and lipase, were used to report on the interfacial stabilization provided to proteins in emulsion ' and elastomeric environments, respectively. 1

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Experimental Reagents The surfactant DC3225C was obtained from Dow Corning; decamethylpentacyclopentasiloxane (D ), octamethylcyclotetrasiloxane (D ), silanol-terminated poly(dimethylsiloxane) ((HO-SiMe (OSiMe )nOSiMe OH, η * 484), 2000 cSt, 36000 MW), dibutyltin dilaurate, tetraethyl orthosilicate (TEOS), polyethylene oxide) (PEO) (2000 MW), sodium hydride (60% dispersed in oil), Celite, allyl bromide, triethoxysilane, lauric acid, dimethylsulfoxide (DMSO), and octanol were purchased from Aidrich. Vinylterminated polydimethylsiloxane (1000 cSt and 2-3 cSt) and platinum divinyltetramethyldisiloxane complex (3-5% platinum concentration, Karstedt's catalyst) in vinyl-terminated polydimethylsiloxane were obtained from Gelest. 5

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258 tyA^dimethylformamide (DMF), isooctane, dichloromethane, acetone, and cyclohexane were purchased from Caledon. Human serum albumin (HSA) 9699% purity, blue dextran, ct-chymotrypsin, lipase from C. rugosa (EC 3.1.1.3), lysozyme, protein assay kits (Lowry method) and Sephadex G-25 were purchased from Sigma. 5-(Bromomethyl)fluorescein (5-BMF) was supplied by Molecular Probes and used to label HSA. TES-PDMS (bis(triethoxyethyl)dimethylsilicone), TES-PEO (bis(triethoxy-silylpropyl) polyethylene oxide) including MW 2000, TES-PEO2000), and TES-mPEO 350 (triethoxysilylpropyl, methyl poly(ethylene oxide)), and 5BMF-HSA were prepared using literature procedures. 17

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Emulsion Formulation Procedure: Example using 5-BMF-LabeIled HSA and TES-PDMS 17

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Emulsions were formulated using established procedures. HSA (0.612 g) and 5-BMF-HSA (1.0 mL, 1.99 χ 10" mg/mL) were dissolved in 1.0 M Tris-HCl buffer (30.0 mL, pH 7.8). Triethoxysilylethyl-terminated PDMS (4.14 g)) was dissolved in D (16.1 g, continuous phase) in the mixing vessel. The aqueous solution was added to the silicone oil phase in a continuous, drop-wise manner over a period of 2 h under turbulent mixing conditions at 2780 rpm using a Caframo BDC6015 mixer. The emulsion was allowed to mix for an additional 2 h and stored in a sealed container at ambient temperature. Emulsions were imaged using a Zeiss LSM 510 confocal microscope. 4

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Elastomeric Composite Preparation: Example Lipase in Silicone

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Lipase (100 mg) and, optionally, water were sonicated for 1-2 min in CH C1 (~ 1 mL) then added promptly to a vial containing silanol-terminated polydimethylsiloxane (HO-PDMS, 2.85 g), and crosslinker (TEOS, 0.15 g). TES-mPEO 350 (-350 MW) was optionally included (0.3 g in CH C1 ). The mixture was mixed vigorously with a spatula and then dibutyltin dilaurate catalyst (0.05 g, 0.08 mmol) was added. Mixing was continued for 4-5 min after which the mixture was poured onto small Petri dish (60 mm χ 15 mm) and left uncovered for ca. 30 min, then covered and kept at room temperature to cure overnight. Cylindrical pellets of ca. 6.0 mm diameter and 1.8-2.2 mm thickness were cut from the cured film and, after washing with solvents (acetone, water, acetone, cyclohexane, then acetone) and air drying, granules of the composite were prepared by grinding the composite using a mortar. After swelling in isooctane, enzymatic activity was established using transesterification, as discussed in the text. 2

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Results


Emulsions Human Serum Albumin and a-Chymotrypsin Two different w/o emulsion systems were used to study the nature of protein/silicone interactions at an aqueous/oil interface: one employing the use of a silicone surfactant with terminal triethoxysilyl moieties (TES-PDMS, Figure 1 A), and the second with a commercially available silicone surfactant possessing pendant poly(ethylene oxide) (PEO) groups (commercially available DC3225C). Emulsions were formulated by dissolving the silicone surfactant of interest in octamethylcyclotetrasiloxane (D ), and subsequently adding drop-wise the aqueous phase, which optionally contains proteins, under dual-blade, turbulent mixing conditions. With the functional, poorer surfactant TES-PDMS, the presence of a surface active protein, human serum albumin (HSA), was required in order to form a stable w/o emulsion. ' Water-in-oil emulsions were formed with both surfactants, with protein dissolved in discrete water domains suspended in bulk silicone oil. ' Confocal microscopy experiments performed on w/o emulsions containing fluorescently labelled proteins demonstrated that the protein preferentially resided at the water/silicone oil interface, indicating that contact between the biomolecule and the silicone oil was possible (Figure IB). The natural fluorescence of the single tryptophan residue provides a mechanism to assess the unfolding of HSA. During the unfolding processes, the fluorescence maximum is first blue and then red shifted. ' It was shown that comparable rates of unfolding were observed for HSA in buffer, and in emulsions stabilized with DC3225C and TES-PDMS, respectively: all emulsions were blue shifted by day 10. Despite the fact that the protein molecules are subjected to a great deal of mechanical stress during the emulsification process (6 h at 2780 rpm), and that the proteins are encountering silicone molecules at the emulsion interface, the proteins are protected by surfactants from silicone contact. ' In some cases, emulsion stability correlates with HSA unfolding, for example, TES-PDMS emulsions were stable for 1-6 months, at which point the HSA was completely denatured. With the better surfactant DC3225C, enzyme unfolding plays no role in emulsion stability: stable emulsions are readily prepared using either native or denatured proteins, or no proteins at all. The exposure of an aqueous solution of chymotrypsin in buffer to silicone oil (octamethylcyclotetrasiloxane, (Me SiO) , D ), in the absence of a surfactant, led to a rapid loss of enzymatic activity (85% loss). After exposure to D , the enzyme was unable to transesterify ethylJV-acetyl-Z,-phenylalanate with butanol. Under similar conditions but with addition of the surfactant DC3225C, comparable enzyme activities between the control and the emulsified proteins were observed (Figure 2A). Activity remained despite the significant mechanical energy required to prepare and break the emulsion, and the possibility of contact with silicone during mixing. This is surprising given thefragilityof the enzyme. ' 4

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Figure 1. A: Structures ofsilicone surfactants; B: 5-BMF-labelled HSA shown as a corona at the interface of a w/o emulsion


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261 These observations, based on two distinctly different proteins, suggest that a strong interaction exists between the surfactant and protein that either protects the protein from contact with unfunctionalized silicone oil during mixing, or which stabilizes protein tertiary structure.


Silicone Elastomers: Chymotrypsin and Lipase Silicone-chymotrypsin elastomers were prepared using room temperature vulcanization (RTV) with the optional inclusion of functional PEO (e.g., MeO(CH CH 0)„(CH2)3Si(OEt)3) using silanol terminated silicones (HO(SiMe 0) H, ca. 36000 MW) and TEOS (Si(OEt) ), catalyzed by dibutyltin dilaurate in the presence of moisture (RTV conditions ). In isooctane, optimum enzyme activity occurred when the enzyme was ultrasonicated in the presence of small quantities of water (no water, no sonication, activity = 0.001 mg/h per mg enzyme; sonicated with 0.2% water (a -1.0), activity = 0.014 mg/h per mg of enzyme). At the same low water concentrations, the activity of chymotrypsin in D was approximately the same as in isooctane, a result quite different from that noted above in excess water. The activity of the enzyme degraded over time in isooctane, whereas in the PEO-containing elastomer the activity was preserved over several consecutive reaction cycles (Figure 2B). 2

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Figure 2. A. Enzyme activity of a-chymotrypsin A. extractedfrom a w/o emulsion vs control ; B. In plain silicone elastomers that optionally contain mono-functional PEO (TES-mPEO350). 31

Lipase is known to be generally tolerant of hydrophobic media and, in nature, generally resides at cell membranes. ' The enzymatic activity of lipase in hydrophobic media indicated that lipase has a much higher enzymatic activity 27 28



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Figure 3. A. Activity offree lipase (0.52mg) in isooctane and D5 in the presence of different amounts of water. B. Effect of includingfunctional PEO in the elastomers (8%TES-PEO2000) on the activity of the entrapped lipase, measured by catalyzed esterification reaction. C. Comparison of enzyme activity as a function of media.

in D , compared to isooctane (Figure 3A), as judged by its ability to convert octanol and lauric acid into octyl dodecanoate at different water concentrations. Silicone elastomers with and without functional PEO were prepared from silanol terminated silicones as noted above with incorporation of lipase as a powder or, when functional PEO was added to the elastomer, as a slurry. After curing, elastomer disks (6 mm diameter, 2 mm thickness) were cut and examined as catalysts for ester formation from octanol and lauric acid over repeated cycles. Activity is expressed relative to the free enzyme activity (production of ester as determined by GC) in isooctane solution (taken as a specific activity of 1.0). 5

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263 Results showed that lipase activities followed the order: silicone elastomer, no PEO > elastomer with PEO > lipase powder. Thus, the presence of hydrophilic materials, with the exception of minimal amounts of water, were counterproductive with respect to lipase activity (Figure 3B).


Discussion The behavior of three constitutionally different proteins, with vastly different characteristics, has been examined on contact with silicones. It is apparent that all three proteins can be made to be reasonably compatible with silicones provided that the functionality grafted to the silicone, and the concentration of water, are carefully controlled. Three control elements were used to manipulate protein stability in a silicone environment: local viscosity, water concentration and the presence of PEO. Lipase, known for its ability to withstand hydrophobic media, was found to be active in silicone oil, and more active in the less mobile elastomer. It is possible that an interface is established between silicone and protein when the elastomer cures, which restricts the ability of the enzyme to unfold. In such a case, the active site of the enzyme can anchor at the hydrophobic interface, and be held in an active conformation by the locally high viscosity. The incorporation of hydrophilic media such as PEO in the elastomer reduces the local hydrophobicity of the interface and, consequently, the activity of the enzyme. For this enzyme, the local water concentration was less significant than for other proteins. HSA unfolds readily at a silicone/water interface. However, with the addition of non-ionic surfactants, the protein is protected from unfolding even though it lies at the PEO surfactant interface, as shown by fluorescence experiments. PEO may be increasing the local viscosity, acting as an osmolyte, or simply insulating the HSA from the silicone. Such protective roles are exploited therapeutically for protein drugs. A different role must be ascribed to TES-PDMS, which led to a reduction in the rate of HSA unfolding within the emulsion droplets, and to the ability to create and stabilize a silicone/water interface: an emulsion of water and D could not be formed from either HSA or TES-PDMS on their own. Although the HSA is a constituent of the interface, it unfolds at a rate similar to that of the control. Of more interest is the correlation between emulsion stability and protein HSA folding in these emulsions, which suggests the HSA/TES-PDMS interaction may result from a binding event which becomes disfavored as the protein starts to unfold. Such a possibility provides an alternative method to release the contents of the water droplet through controlled HSA denaturation. ct-Chymotrypsin denatures at silicone/water interfaces. When a surfactant such as DC3225 was present, the enzyme sat at the emulsion interface and, as 27,28

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264 with HSA, was protected from exposure to the silicone as evidenced by its enzymatic activity, which was comparable to control in spite of the mechanical energy used to make and break the emulsion. On one hand, there is a driving force for proteins to adsorb at interfaces. On the other hand, PEO-interfaces are typically protein repellent. The balance of these two can account for the observed enzymatic behavior in w/o emulsions. A similar situation arises with chymotrypsin in silicone elastomers doped with PEO. In this case, the enzyme must be PEO coated to avoid the denaturation that normally occurs on contact with silicone. The rate of denaturation in elastomers is retarded by higher viscosity, and by the very low availability of water; compare this with rapid denaturation in emulsions, when the protein is swollen with water and internally mobile. Water can be used to regulate enzyme activity in these systems; small amounts are needed for catalytic activity, but increased degrees offreedomfromtoo much water lead to unfolding and inactivation. By controlling the mobility of the interface, and the presence or absence of hydrophilic surface active materials, it was possible to stabilize three proteins of widely varying structure, function, and properties in hydrophobic silicone oil or elastomer domains. It is, therefore, likely that silicone-based environments can similarly be formulated for other proteins, which opens the possibility to use silicone-, rather than organic-based, materials, as biosensors, immobilized enzymes, and drug delivery vehicles. 17

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Conclusions Silicones are exceptionally hydrophobic materials that interact with enzymes, mainly through hydrophobic interactions, frequently causing denaturation leading to loss of enzymatic activity. Enzymes can be rendered stable and, in some cases, more active in silicone oils or elastomers by using the presence of non-ionic surfactants and water: specific formulations need to be developed for different enzymes.

Acknowledgements We thank the Natural Sciences and Engineering Research Council of Canada for financial support of this work.

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Proteins at Silicone Interfaces - American Chemical Society

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