Stability & activity of therapeutic biomolecules in ionic liquids

Interest in ionic liquids (ILs) has grown with its evolution now spanning three generations: (1) ILs with tunable physical properties; (2) ILs with tunable chemical properties; and (3) ILs with tunable biological properties. A growing number of biological applications using ILs are underway as a replacement solvent for synthesis, for use as a biological constituent and for preserving and enhancing the functionality of biomolecular species. Therapeutic biomolecules play a significant role in the medical field and in the research field, with many being trialed and developed for various applications; primarily as the name suggests for therapeutic uses. The storage and stability of therapeutic biomolecules has long been an issue. It is now among common knowledge to store therapeutic biomolecules at low temperatures ranging from 4°C to -196°C, depending on the concentrations in use. Ideally the freeze thaw cycles are also limited to maintain stability and activity. It is also commonly kept on ice whilst in use in a laboratory space. Although the use of refrigeration and ice in laboratories and hospitals may not be an issue, in third world countries this is still a significant problem. The transportation and storage of such therapeutic molecules can be problematic, hence the storage, stability and activity to be maintained at room temperature is vital in sterile and non-sterile conditions. The focus of this thesis is to preserve the structure and activity of biomolecules using choline dihydrogen phosphate (CDHP) buffered ionic liquid (BIL). Choline is an essential nutrient in the human body, and hence a biocompatible IL is formed using choline as the cation. Choline is currently FDA approved for pharmaceutical excipients making it a favorable selection for a constituent of the ionic liquid. In light of current literature reports the stability of the biomolecules are important and is assessed via thermal stability, structural integrity and biological stability and activity. The storage of therapeutic biomolecules in CDHP-BIL was compared to phosphate buffered saline (PBS) a common storage buffer for biomolecular species. Studies were conducted for prolonged periods in conjunction with enzymatic degradation and characterized using a number of techniques including assays, gel electrophoresis, circular dichroism, UV-Vis spectrometry, flow cytometry and confocal microscopy. Three therapeutic biomolecules have been examined for this project: 1) Small interfering ribonucleic acid (siRNA), 2) Plasmid deoxyribonucliec Acid (pDNA) and 3) Monoclonal antibodies (mAbs) Each chapter of this thesis is dedicated to each therapeutic biomolecule mentioned above. Chapter 2 reports the stabilization and retaining of biological activity of siRNA. Chapter 3, demonstrates that the stability of nucleic acids is not limited to siRNA, proving that CDHP-BIL can impede enzymatic degradation and the expression of pDNA can be enhanced. As a continuation of bimolecular therapeutic we moved on to proteins in Chapter 4, reporting inhibition of proteinase K when mAbs are stored in BILs. Finally this thesis envisages the future directions and challenges for the long term storage and stabilization of therapeutic biomolecules at room temperature. The simple manner of being able to store therapeutic molecules in a buffer that can enhance the properties of the biomolecules whilst prolonging its shelf life, is extraordinary. The applications of this study are far and wide once integrated in the medical field as it has the potential to be a new green bio-buffer.