Ionic liquids are being intensely studied as promising media for the stabilization of proteins and other biomolecules. Choline dihydrogen phosphate (CDHP) has been identified as one of the most promising candidates for this application. In this work we have probed in more detail the effects that CDHP may have on the thermodynamics, structure, and stability of proteins, including one of therapeutic interest. Microcalorimetry and circular dichroism spectropolarimetry (CD) were used to assess the thermal stability of protein solutions in CDHP/water mixtures at various concentrations. Increasing thermal stability of lysozyme and interleukin-2 in proportion to CDHP concentration was observed. Isothermal titration calorimetry (ITC) was used to quantify binding interactions, and indicate that the mechanism for stability does not appear to be dependent upon CDHP binding to protein. CD and small angle X-ray scattering (SAXS) analyses were used to probe for structural changes due to the presence of CDHP. SAXS indicates charge effects on the surface of the protein play a role in protein stability in ionic liquids, and no significant alteration of the overall tertiary conformation of lysozyme was observed at 25 °C. However, after incubation at 37 °C or at higher concentrations of CDHP, small changes in protein structure were seen. Effects on protein activity were monitored using turbidity assays, and CDHP decreases protein activity but does not eliminate it. Protein solubility was also monitored using a turbidity assay and was found to be inversely proportional to the concentration of CDHP in solution.

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 dihydrogen phosphate (CDHP) is an ionic liquid reported to increase thermal stability of model proteins. The current work investigated CDHP effect on structural integrity and biological activity of recombinant human interleukin-2 (rhIL-2), a therapeutic protein used for treating advanced melanoma. In vitro CDHP biocompatibility was also evaluated using primary cell cultures, or B16-F10 cell line, chronically exposed to the ionic liquid. Formulation of rhIL-2 in an aqueous 680mM CDHP pH 7.4 solution resulted in a 12.5°C increase in the Tm of rhIL-2 compared to a basic buffer formulation, and provided conformational rhIL-2 stabilization when the solution was heated to 23.3°C above the Tm. CDHP solutions (≤80mM), exhibited no cytotoxic activity toward primary splenocytes or B16-F10 cells in culture. However, a 10-fold loss in biological activity was observed when rhIL-2 was used in a 30mM CDHP aqueous solution with NaHCO3 (pH≥7.2) compared to controls without CDHP. While increased Tm is associated with a diminished rhIL-2 biological activity, the therapeutic protein remains structurally intact and functional.

A low-cost pulsed laser is used in conjunction with a homebuilt laser confocal-scanning epifluorescence microscope having submicron lateral and axial spatial resolution to determine cytoplasmic viscosity at specific intracytoplasmic locations in J774 mouse macrophage cells. Time-dependent fluorescence anisotropy measurements are made at each location and global deconvolution techniques are used to determine rotational correlation times. These rotational correlation times are related to the hydrated volume of 8-hydroxyperene-1,3,6-trisulfonic acid (HPTS) to calculate viscosity at specific points inside the cell. In the cytoplasmic areas measured, rotational correlation times of HPTS ranged from 0.186 ns to 0.411 ns, corresponding to viscosities ranging from 1.00 +/- 0.03 cP to 2.21+/- 0.05 cP.