Final Report Summary - NANOEP (Single Cell Electroporation and DNA dynamics: from bulk to micro/nanofluidics) Publishable Summary:Proposal title: Single Cell Electroporation and DNA dynamics: from bulk to micro/nanofluidicsProposal Acronym: NanoEPSecond Reporting Period: 01/03/2014-31/03/2017The NanoEP project is an EC-FP7 funded Marie Curie Integration Grant (CIG) to support Dr. Boukany (as a young researcher) when integrating at the Delft University of Technology in the Netherlands, (which has been carried at the Chemical Engineering Department). In this MC-CIG, we have focused on the obtaining in depth knowledge of molecular mechanism underlying electroporation of cell membrane by using single cell and molecule techniques. This project started effectively on 01/O4/2013 and has been completed on 31/03/2017. This final report summarizes a description of the objectives, work performed and the main results achieved (01/03/2013 to31/03/2017). The main aim of gene therapy is to deliver therapeutic genes into cells to alter their function. Safe and efficient intracellular delivery of DNAs and drugs holds a great promise for treating diseases such as cancer. Viruses are the most efficient vectors for gene transfer to target cell, but their application in humans raises many safety concerns. In recent years, it is clear that delivering naked DNA into living cells for gene therapy applications is the best safe alternative to conventional viral vectors and chemical delivery methods. Cells are however surrounded by a selectively permeable lipid bilayer that separates the cell interior from the external medium. The membrane of cell is impermeable to large biomolecules such as plasmids and DNAs. Electroporation, a method that enhances cell membranes permeability by subjecting them to electric field pulses is foreseen today as a promising technique that enhances intracellular gene delivery; yet, it is currently still a crude and inefficient technique. Our lack of understanding of cell electro-pore formation and DNA electro-transfer prevents us from optimizing its use: As it stands today, the technology still suffers from too low transfection efficiency, random uptake and excessive cell damage.Tasks Performed and Main Results Achieved The main aim of NanoEP is to understand and control the transport of the biomolecules such as DNA in electroporation process such that stable and safe and efficient gene transfection can be achieved. In this project, we have developed single molecule techniques with high spatio-temporal resolution to investigate the process of electroporation at different length (from bulk to micro/nano) and time scale (ranging from microseconds to hours). In addition, these unique tools have been integrated with micro/nano-fluidics to study the electroporation and electro-kinetic flow of DNA at micro/nano-scale. To achieve this a number of subsidiary objectives, need to be achieved. These are listed below:-To identify the mechanism of electroporation in bulk and micro/nano-fluidics-To unravel the dynamics of electro-pore formation and resealing -To understand the electro-transfer of DNA into nucleusMain results from tasks performed in the reporting period we:- developed novel and unique nano-platform called nano-channel electroporation to deliver precise amount of cargos intro living cells-integrated nep-devices with single molecule techniques to understand the molecular and sub-cellular response in the presence of external forces-created new micro/nano-fluidic devices to manipulate the cellular membranes for understanding the electroporation at the microscopic level -unraveled the molecular process behind electroporation by using simplified cell models and microscopy techniquesThere is currently great interest in the development of new methods for safe efficient of DNA into living cells, both from a scientific and from a social point.Understanding the principles of electroporation and DNA electro-transfer into living cells is of high importance for the development of novel strategies of non-viral gene delivery in both fundamental biological and biomedical applications with enormous societal impact. Prospects of Research Career and Integration of the MC Fellow:The aim of this MC-CIG to support young researcher when integrating at research activities are now fully underway. Several training events, workshops, internal and external collaborations have occurred, when this project commenced. Furthermore, PI has set up new collaborations through these events at national and international level. This project had met its objectives and notable results are already being produced through research carried out in Boukany’s lab at Delft University of Technology. MC fellow has presented his scientific works in several international/national conferences and workshops (Biophysical Society meeting, American Physical Society, Congress of electroporation, Society of Rheology, IEEE meetings, Fundamental research on matter (FOM), etc) and shared his knowledge with researchers in the Europe and the other worlds. Therefore, the MC-CIG was of benefit to the future career of the fellow who worked as an assistant professor in the Chemical Engineering Department at Delft University of Technology to strengthen his network and visibility nationally and internationally. Furthermore, MC fellow has been awarded an ERC-STG to consolidate his own research team (2 PhD students+2Post-Docs) and to start conducting independent research on electroporation by performing expedients at single cell/molecule level. This CIG grant allows PI also to initiate new collaborations with other scientists in the field of non-viral gene therapy. In addition, recently MC fellow has has been awarded a Van Gogh travel grant by Nuffic to deepen his collaboration with Prof. Mounir Tarek to investigate electroporation of cell membranes at the molecular level (by both MD simulations and experimental approaches). The grant allows people from both groups to spend some time at each other's institutes. New collaborations with other groups have resulted in several journal publications (which has been published in Small and Advanced Healthcare Materials, etc). Two articles have been featured and highlighted as a cover story. The MC fellow has a public website (http://cheme.nl/ppe/people/boukany.shtml) where already all the knowledge and the important achievements of this project have been announced in News section.Therefore, the Career Integration Grant has fulfilled the objective to contribute to the integration of Dr. Boukany and his career development as independent researcher after joining as a faculty of chemical engineering.
