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

Abstract

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 80 wt % 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<choline saccharinate<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.

How ionic liquids can help to stabilize native proteins

“…In biomedical applications biocompatibility is mandatory. 25,26 With regard to biocompatibility, ILs cover a wide range from food-grade quality to highly toxic compounds. Strategies for designing biocompatible ILs can build upon ions that exist in nature.…”

“…A prominent example is choline (V), which is a micronutrient. 25,26 Nature also offers biocompatible anions such as saccharinate, citrate or lactate. Elliott et al 25 have conjectured that in future the need for biocompatibility will shift interest from the ILs in use toward ''greener'' species.…”

“…In biochemical applications the power of ILs is largely increased by the possibility to design biocompatibility into their ions. 25,26 Among a plethora of biochemical applications of ILs, [21][22][23][24] enhancements of the thermal and functional stability of proteins [21][22][23][24][27][28][29][30][31][32][33] open intriguing prospects for steering biotransformations. It is also possible to use ILs for destabilizing proteins systematically.…”

Ionic Liquid Gel‐Assisted Electrodes for Long‐Term Cutaneous Recordings

“…ILs have been used in many biological applications, including as solvents that enhance the stability of proteins. [ 17 ] Although several IL families showing low cytotoxicity have been identifi ed, [18][19][20] the establishment of general design rules for IL biocompatibility is the focus of ongoing work. ILs can be polymerized to yield gels, which are especially appealing for use as a quasi-solid-state electrolytes.…”

Hydrophilic Ionic Liquids as Ingredients of Gel-Based Dermal Formulations

“…Furthermore, the ILs can be used as solvents for pharmaceutical components (30) and choline salts can be useful for stabilization of protein-based APIs in the formulation (31 Formulations that included SEPINEO™ P 600 resulted in high-viscosity gels or emulsion-gels. Addition of cetylstearyl alcohol as an additional thickener led to a slight increase in viscosity, in comparison to the formulation without cetylstearyl alcohol, due to the formation of a liquid crystalline phase (32).…”