Search Results (6)

This work unfolds functionalized ABSs composed of FILs ([C₂C₁Im][C₄F₉SO₃] and [${\mathrm{N}}_{1112\left(\mathrm{OH}\right)}$][C₄F₉SO₃]), mere fluoro-containing ILs ([C₂C₁Im][CF₃SO₃] and [C₄C₁Im][CF₃SO₃]), known globular protein stabilizers (sucrose and [${\mathrm{N}}_{1112\left(\mathrm{OH}\right)}$][C₄F₉SO₃]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K₃PO₄). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs’ biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C₂C₁Im][C₄F₉SO₃] + 2 %wt K₃PO₄ and 30 %wt [C₂C₁Im][C₄F₉SO₃] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity. Full article

Interferon alpha-2b (IFN-$\alpha$2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-$\alpha$2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C${}_2$C${}_1$Im][C${}_4$F${}_9$SO${}_3$] and [N${}_{1112\left(\mathrm{OH}\right)}$][C${}_4$F${}_9$SO${}_3$]) vs. mere fluoro-containing IL ([C${}_4$C${}_1$Im][CF${}_3$SO${}_3$]), in combination with sucrose or [N${}_{1112\left(\mathrm{OH}\right)}$][H${}_2$PO${}_4$] (well-known globular protein stabilizers), or high-charge-density salt K${}_3$PO${}_4$ were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-$\alpha$2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-$\alpha$2b high in the enriched IFN-$\alpha$2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-$\alpha$2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-$\alpha$2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics. Full article

Proteins are inherently unstable, which limits their use as therapeutic agents. However, the use of biocompatible cosolvents or surfactants can help to circumvent this problem through the stabilization of intramolecular and solvent-mediated interactions. Ionic liquids (ILs) have been known to act as cosolvents or surface-active compounds. In the presence of proteins, ILs can have a beneficial effect on their refolding, shelf life, stability, and enzymatic activities. In the work described herein, we used small-angle X-ray scattering (SAXS) to monitor the aggregation of different concentrations of ILs with protein models, lysozyme (Lys) and bovine serum albumin (BSA), and fluorescence microscopy to assess micelle formation of fluorinated ILs (FILs) with Lys. Furthermore, coarse-grained molecular dynamics (CG-MD) simulations provided a better understanding of Lys–FIL interactions. The results showed that the proteins maintain their globular structures in the presence of FILs, with signs of partial unfolding for Lys and compaction for BSA with increased flexibility at higher FIL concentrations. Lys was encapsulated by FIL, thus reinforcing the potential of ILs to be used in the formulation of protein-based pharmaceuticals. Full article

Phenylketonuria (PKU) is an autosomal recessive disease caused by deficient activity of human phenylalanine hydroxylase (hPAH), which can lead to neurologic impairments in untreated patients. Although some therapies are already available for PKU, these are not without drawbacks. Enzyme-replacement therapy through the delivery of functional hPAH could be a promising strategy. In this work, biophysical methods were used to evaluate the potential of [N_1112(OH)][C₄F₉SO₃], a biocompatible fluorinated ionic liquid (FIL), as a delivery system of hPAH. The results herein presented show that [N_1112(OH)][C₄F₉SO₃] spontaneously forms micelles in a solution that can encapsulate hPAH. This FIL has no significant effect on the secondary structure of hPAH and is able to increase its enzymatic activity, despite the negative impact on protein thermostability. The influence of [N_1112(OH)][C₄F₉SO₃] on the complex oligomerization equilibrium of hPAH was also assessed. Full article

Proteins are bioactive compounds with high potential to be applied in the biopharmaceutical industry, food science and as biocatalysts. However, protein stability is very difficult to maintain outside of the native environment, which hinders their applications. Fluorinated ionic liquids (FILs) are a promising family of surface-active ionic liquids (SAILs) that have an amphiphilic behavior and the ability to self-aggregate in aqueous solutions by the formation of colloidal systems. In this work, the protein lysozyme was selected to infer on the influence of FILs in its stability and activity. Then, the cytotoxicity of FILs was determined to evaluate their biocompatibility, concluding that the selected compounds have neglected cytotoxicity. Therefore, UV–visible spectroscopy was used to infer the FIL-lysozyme interactions, concluding that the predominant interaction is the encapsulation of the lysozyme by FILs. The encapsulation efficiency was also tested, which highly depends on the concentration and anion of FIL. Finally, the bioactivity and thermal stability of lysozyme were evaluated, and the encapsulated lysozyme keeps its activity and thermal stability, concluding that FILs can be a potential stabilizer to be used in protein-based delivery systems. Full article

The task-specific design of ionic liquids (ILs) has emerged in several industrial and pharmaceutical applications. The family of ILs with fluorine tags equal to or longer than four carbon atoms, the fluorinated ionic liquids (FILs), combine the best properties of ILs with the ones of perfluorinated compounds, and are being designed for several specific purposes. In the pharmaceutical field, there is an urgency to search for novel antibacterial agents to overcome problems associated to antimicrobial resistances. Then, the main purpose of this work is to evaluate the environmental impact and the ability of FILs to be used as antibacterial agents against Pseudomonas stutzeri bacteria. Beyond its rare pathogenicity, these bacteria are also used as a bioremediation agent to treat several contamination sites. Then, it is important to determine which FILs have antibacterial properties, and which do not impact the bacterial growth. The biocompatibility of FILs was also evaluated through their hemolytic activity and represent a step forward the application of FILs in pharmaceutical applications. The results proved that high concentrations of FILs can have a reduced ecotoxicity and a high biocompatibility. [C₈C₁Im][CF₃SO₃] was identified as the most promising compound to be used as an antibacterial agent since it prevents the growth of bacteria at concentrations compatible with the red blood cells’ viability. Full article