Periodic Reporting for period 1 - DUNE (Deep eutectic solvents for membrane transport of nucleic acids)
Reporting period: 2023-09-01 to 2025-08-31
The Action DUNE – Deep Eutectic Solvents for Membrane Transport of Nucleic Acids looks at how solvent engineering can be exploited to stabilise nucleic acids and transport those across biological membranes. The delivery of genetic material as therapeutics constitutes one of the most promising approaches to treat emergent pathogens and important diseases. However, the current technologies are threatened by the low stability of the nucleic acids, the limited efficacy of transport across cell membranes, and the relatively high toxicity of the formulations. DUNE aimed to create a groundbreaking approach for the development of deep eutectic solvents (DESs) as a formulation platform for the preservation and cytosolic delivery of nucleic acids. DESs are non-aqueous solvents formed through mixing simple organic molecules with the capacity to solubilise and stabilise biomolecules. The large number of possible combinations of precursors for the preparation of DESs yields a tailorable platform, in which the properties of the solvent can be optimised for the functional stabilisation of nucleic acids. Potentially, this tailorable character can be exploited to develop suitable environments for nucleic acids with the capacity to transport them across biological membranes.
Initially, our studies explored the capacity of anhydrous DESs with varied composition to solubilise nucleic acids. Using spectroscopy and scattering methods, these investigations aimed at deciphering the conformational landscape and colloidal stability of the biomolecules as a function of solvent properties. Then, the possibility of including lipids in the formulation was explored. Finally, the transfecting capacity and toxicity of the DES-lipid-mRNA formulation was investigated in model cell lines.
Compositionally optimised DESs demonstrated the capacity to solubilise biomolecular cargoes (e.g. peptides, nucleic acids) in the absence of water. The biomolecules adopted similar conformations in DESs to those observed in aqueous buffers, thus potentially preserving also their function. When placed in biological environment, the formulated model peptides could be transported through specific DES-cargo and DES-membrane interactions without the need of specific transfecting agents. The formation of a lipid-DES nanocomposite resulted in the stabilisation of nucleic acids in the absence of water. Notably, the formulation resulted in cell transfection with similar efficacies, but reduced toxicity, compared to those observed in water.