Novi lipidni modelni sistemi za določitev mehanizmov elektroporacije (Slovene)

The aim of the proposed project is to design a novel delivery system for various biologically active substances by encapsulating them in artificial liposomes made of archaeal lipids, delivering the liposomes into cells and then controlling the substance release into the cytosol by nanosecond electroporation. In addition, the proposed project will provide improved numerical models of electroporation at the level of membrane, cell and tissue. All this will allow for a better and more efficient treatment planning in electrochemotherapy, tissue ablation by irreversible electroporation, efficient and safe gene electrotransfer for genetherapy, and DNA vaccination.   Clinical benefits of using liposomal formulations have stimulated research and quest for new liposomal varieties. Liposomes prepared from archaeal ether lipids (archaeosomes) have been found to be chemically and physically considerably more stable than conventional liposomes. As such, archaeosomes should also be more convenient for delivery into cells than conventional liposomes and should remain stable inside the cells for longer time.   In the past few years, manipulation of cell organelle membranes has become accessible by the use of extremely strong and short (tens to hundreds of nanoseconds) electric pulses. This promising method is termed nanosecond electroporation and enables e.g. electroporation of postendocytotic vesicles, leading to release of vesicle content into the cytosol. We are planning to use this new method to electroporate the internalized liposomes pre-loaded with drugs.   Although the use of electroporation (including nanosecond electroporation) is increasing rapidly, the mechanisms and the related processes, such as transmembrane transport, are still poorly understood. It is therefore the aim of this project to gain new understanding of the fundamental biological and physical processes of electroporation and of the mechanisms involved in electroporation-mediated molecular transport across the plasma or liposome membrane. This requires the knowledge of the electrical properties of lipid bilayers, cells and tissues, and the interactions between the external electric field and the transport across the cell membrane. To address these issues, an interdisciplinary approach will be used, integrating experiments and numerical modeling on different levels of biological complexity, from simple planar lipid bilayers, via vesicles and cells to tissues (see the attachment figure.pdf).   New lipid systems will be used, composed of very different lipids, such as pure archaeal or mixed lipids with added cholesterol and proteins, forming planar lipid bilayers and vesicles. These different systems will allow full characterization of membrane composition and its biophysical properties. Experiments on these systems and molecular dynamics simulations will be used to understand the mechanisms of electroporation and related transport at the molecular level (e.g. pore formation, pore resealing, pore density, pore size distribution, transmembrane transport). The parameters of electroporation (including nanosecond electroporation) and new knowledge about electroporation and transport obtained at the level of planar lipid bilayers will than be tested on cells in vitro, and finally in tissues in vivo. At the same time the model parameters obtained from atomistic molecular dynamics simulations and experiments on planar lipid bilayers and vesicles will be used to refine the finite element models of cell and tissue electroporation. These models will have predictive power and will be used to optimize the pulse parameters for successful electroporation of internalized liposomes and controlled release of their content. The knowledge and understanding obtained within this project will allow for a better and more efficient treatment planning in electrochemotherapy, tissue ablation by irreversible electroporation, efficient and safe gene electrotransfer for genetherapy, and DNA vaccina

We constructed molecular dynamic models of archaeal lipids, which have not been described in the literature before. The archaeal lipids have special structure. Using molecular dynamic simulations, we investigated the effect of special moieties to the stability of bilayers. The results showed high stability of the archaeal lipid bilayers under electric field. The deformations are different than in regular lipid membranes. This explains why big molecules do not cross the archaeal lipid bilayer. This research improves the understanding of the relation between lipid composition and stability. We improved the growth of A. pernix and assure the biomass for research needs. The price of archaeal lipids is namely very high. We develop production of archaeal lipids, archaeosomes, which were loaded with substances. We measured enthalpy differences and calorimetry and nanoporation of cells. Due to good understanding of properties of archaeal lipids, we can use this knowledge for other technological and applied projects. New knowledge can be also used to develop the technology improvements (biomass production, nanosecond pulse generators…).

The project provided us with better understanding of fundamental mechanisms of electroporation. In spite of broad use of electroporation detailed molecular mechanisms are not yet known. We established fruitfull collaboration between different research groups and intendsified existing by focusing on the electroporation of biological membranes. We established new collaborations with the Faculty of Chemistry and Chemical Technology (University of Ljubljana) and further intensified collaboration with Équipe Théorie Simulations et Modélisation (Université de Lorraine, Francija). The electron density profiles of archaeal lipid vesicles and their mixtures with DPPC were measured at The Faculty of Chemistry and Chemical Technology. The molecular dynamic simulations were done at the Équipe Théorie Simulations et Modélisation, which is one of the leading laboratory in molecular dynamic simulations. This collaboration improves current status of Slovenia in the international research on molecular dynamic simulations.