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Technical Briefs

Biocompatible Ionic Liquids: A New Approach for Stabilizing Proteins in Liquid Formulation

[+] Author and Article Information
Regina M. Vrikkis

Department of Mechanical Engineering, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223rmkemp@uncc.edu

Kevin J. Fraser

Department of Chemistry, Monash University, Wellington, Vic 3800, Australiakevin.fraser@sci.monash.edu.au

Kyoko Fujita

Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho Koganei, Tokyo 184-8588, Japankyokof@cc.taut.az.jp

Douglas R. MacFarlane

Department of Chemistry, Monash University, Wellington, Vic 3800, Australiad.macfarlane@sci.monash.edu.au

Gloria D. Elliott

Department of Mechanical Engineering, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223gdelliot@uncc.edu

J Biomech Eng 131(7), 074514 (Jun 29, 2009) (4 pages) doi:10.1115/1.3156810 History: Received September 01, 2008; Revised May 27, 2009; Published June 29, 2009

Ionic liquids (ILs) have shown excellent promise as both solutes and solvents for stabilizing proteins at room temperature. Because many modern drugs are protein-based, these stabilizing characteristics have great potential to provide advances in the field of liquid formulation of therapeutic proteins. However, before these developments can be translated into clinical solutions it is essential to establish data related to the biocompatibility of these ILs. The current work investigates the cytotoxicity of several ILs that were rationally synthesized from natural biomolecules and compounds that have already been approved as excipients for drug formulations. The effect of choline dihydrogen phosphate (choline dhp), choline saccharinate, and 1-butyl 3-methyl imidazolium lactate (bmim lactate) on the metabolic activity of a mouse macrophage cell line (J774) was assessed using the reduction in resazurin as an indicator of activity and, by extension, viability. Two formulations of lysozyme (10 mg/ml and 100 mg/ml) in 80wt% choline dhp (aq) were prepared and the proteins were evaluated for structural stability immediately following formulation and again at 1 month. Equivalent formulations in 0.1 M Na acetate aqueous buffer were evaluated as controls. A differential scanning microcalorimeter (DSC) was used to evaluate the structural stability on the basis of the unfolding temperature and the enthalpy of unfolding, and a micrococcus lysodiekticus activity test was used to evaluate functional activity. All compounds were found to be relatively benign, with toxicity increasing in the order choline dhp<cholinesaccharinate<bmim lactate. At 1 month lysozyme that had been stored in choline dhp had a higher activity and folded fraction than lysozyme that had been stored in aqueous buffer. These results suggest that biocompatibility and protein stabilization characteristics can be rationally designed into ionic liquids.

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Copyright © 2009 by American Society of Mechanical Engineers
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Figures

Grahic Jump Location
Figure 1

Ions comprising choline dihydrogen phosphate

Grahic Jump Location
Figure 2

The viability of J774 cells was assessed as the normalized response of treated cells to untreated controls as measured by the emission intensity of the metabolic indicator resazurin. Data shown represent the dosage response of cells to (a) choline saccharinate and (b) choline dhp.

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