Selektivna elektroporacija s porazdeljenimi nanoelektrodami

The aim of the project is to systematically explore the phenomenon of membrane electroporation/permeabilization and intracellular drug accumulation and introducing nucleic acids (e.g. DNA) into cell using pulsed electric fields with nanoparticles theoretically (WP1), and experimentally using different nanoparticles (WP2) and demonstrating transmembrane transport, drug accumulation in cells and gene transfection in vitro (WP3). The combined theoretical and experimental approach will enable systematic investigation and understanding of cell membrane-nanoparticles-electric field interaction. Special care will be given to disseminate results of the project to the scientific community through different communication channels and to identify and protect intellectual property if and when deemed necessary (WP4). WP1. Theory and modeling In WP1, we will investigate lipid bilayer interaction with small (e.g. ≤5 nm) gold nanoparticles and membrane modification due to NP near the membrane by atomistic and coarse grain molecular dynamics simulations and of larger NPs (20 and 50 nm) using continuum numerical models using finite element method and distributed element model. The parameters like shape, size and distance from the membrane will be investigated with respect to induced transmembrane voltage, pore density, electric field and charge distribution within the lipid bilayer and its capacitance. The results of models will guide the choice of NPs in WP2 and will be used to design experiments in WP3 in the attempt to validate theoretical predictions using experimental approaches described in WP3. In WP2 we will design, synthesize, functionalize, and conjugate well-defined gold NPs (Figure 4 and 5). The choice and functionalization strategy of NPs to be used in experiments in WP3 will be guided by theoretical predictions and calculations performed within WP1. Specific NPs with high electron microscopy contrast will be examined after being incubated with cells to determine the distance of NPs bound to the plasma membrane and determine membrane thickness. These results will serve to validate results obtained in WP1, and together with experimental results obtained in WP2 will help validating models developed in WP1. In WP 3, we will explore the phenomenon of cell membrane electroporation/permeabilization, intracellular drug accumulation and gene electrotransfer through in vitro experimentation using pulsed electric fields and different NPs identified in WP1 and synthesized and characterized in WP2. We will start by performing experiments on cell suspension mixed with fluorescent dyes that are commonly used for detection of cell permeabilization. Cell membrane permeabilization and survival will be determined after exposure to pulsed electric fields in presence of different NPs. Using optimized pulse parameters, NPs concentration and incubation conditions, we will then deliver cytotoxic molecules and nucleic acids into cells. In WP4 we will protect intellectual property and disseminate achieved results of the project to interested scientific community. Since the interaction of nanoparticles interaction with membranes and enhanced membrane electroporation by NPs can be of wide interest, we will take special care to use various communication channels, such as scientific journals and magazines, conferences, and congresses to disseminate project results. The results will be published in journals Bioelectrochemistry, IEEE Transactions on Biomedical Engineering, Small, Biomaterials, Nanomedicine and presented at the World Congress on Electroporation, European Microscopy Congress, International Microscopy Congress. In order to exchange knowledge and expertise related to enhancing cell permeabilization using NPs, we will prepare a special session at the World congress of Electroporation in 2023, and a training course in the scope of the International scientific workshop and postgraduate course Electroporation based Technologies and Treatment – the postgraduate course and scientific workshop that we organize on an annual basis (www.ebtt.org).