|Selective electroporation by distributed nanoelectrodes (2021-2024)|
|Programme head:||Prof. Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering|
|Funding:||Slovenian Research Agency (ARRS), Slovenia|
The project is funded by the Slovenian Research Agency (ARRS).
- Member of University of Ljubljana
- Selective electroporation by distributed nanoelectrodes
- 1.10.2021 - 30.9.2024
- Amount of financing
- 1.3 FTE
- Research activity
- Engineering sciences and technologies - Systems and cybernetics
- Research Organisation
Electroporation is becoming a widely used method for transiently increasing the permeability of cell membranes by exposing cells or tissues to short and intense electric pulses. Electroporation is especially gaining momentum for local delivery of chemotherapeutic drugs into tumor cells and for intracellular delivery of nucleic acids for various forms of gene therapy. One of the most important advantages of electroporation is its universality, as it can be used to permeabilize the membrane of literally any type of cell. However, this advantage becomes a disadvantage, when we need to selectively electroporate specific cells within a complex tissue arrangement.
The aim of the project is to develop a new protocol which will enable selective electroporation of targeted cells for drug and gene delivery. To this end we will use conductive gold nanoparticles (NP), for which our (and others’) previous work suggests that they can act as distributed nanoelectrodes and locally amplify the applied electric field. To improve membrane electroporation in this way NP is required to be in the vicinity of the cell membrane. These nanoparticles can be functionalized to interact with membranes of specific cells. We thus hypothesize that by combining electroporation with cleverly selected nanoparticles, we can achieve the desired selective electroporation.
Within this project we will test our hypothesis by means of theoretical and experimental approaches and joint efforts of nanomaterial scientists, cell biology experts, and world leading experts on electroporation. The project is organized in four work packages (WP). WP1 is devoted to theoretical understanding of the interconnections between the interactions of nanoparticles with the cell membrane, local electric field amplification, and electroporation. We will develop this understanding by using our expertise and experience on molecular dynamics simulations and finite element modelling. The activities within WP1 are closely linked with WP2, which is devoted to in vitro experiments on systems with increasing biological complexity: planar lipid bilayers, giant plasma membrane-derived vesicles, and different cultured cells. Planar bilayers and giant plasma membrane vesicles will provide and excellent model system for studying the nanoparticle-membrane interaction as both can be used with simple electrolytes in which behavior of functionalized NPs is more predictive than in complex media needed for cell culturing. These experiments will further enable close comparison to the theoretical work in WP1. The use of various cell lines, the widely-studies CHO cells, normal porcine urothelial cells, and human bladder cancer cell lines of different grades, will enable us to characterize the selectivity of electroporation and drug delivery achieved with NPs. We will explore gold NPs by systematically varying their properties: size (5-50 nm), coating (rough or smooth) and surface functionalization. The work in WP1 and WP2 will be upgraded in WP3, which is focused on demonstrating transmembrane transport, drug accumulation and gene transfection in cells in vitro. Finally, WP4 will ensure efficient dissemination of the project’s results, as well as identification and protection of intellectual property.
The project contains numerous innovative and ambitious methodological approaches, which require interdisciplinary expertise. Therefore, the project will be conducted in collaboration among the Faculty of Electrical Engineering and the Faculty of Medicine, both at the University of Ljubljana, and the Department for Material Synthesis at the Jožef Stefan Institute in Ljubljana, Slovenia.
The results of the project will provide the bases for more efficient and selective electroporation by means of NP functionalization. The results of project will also provide new insights in nanoparticles interactions with cell membranes which are of interest to a wider scientific community.
- The phases of the project and their realization
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).