Periodic Reporting for period 1 - OPTIC BIOEM (Toward the comprehension of primary bioelectromagnetic interactions: real time non-linear OPTICal imaging of BIO-samples under ElectroMagnetic exposure)
Berichtszeitraum: 2015-09-01 bis 2017-08-31
Context and overall objectives: In bioelectromagnetics research, one of the major scientific limitations is the lack of hypotheses and experimental corroboration of interaction mechanisms. This issue is extremely pertinent in medical applications of EMFs, where one of the most studied topics is related to the mechanisms of cell membranes permeabilization through pulsed E fields. Beside a first hypothesized mechanism for cell membranes permeabilization mediated by mechanical rearrangement of the membrane phospholipids, the E pulse delivery seems, rather, to alter or promote chemical reactions involving phospholipids peroxidation of cell membranes which, as a consequence, are responsible also for their structural changes. This new hypothesis could have a great impact on the definition and optimization of biological and medical treatments and protocols using pulsed E fields.
Thus, the use of an optical non-linear imaging technique (i.e. CARS) seems extremely suitable to follow and image, with a time resolution of few nanosecond, the dynamics of specifically chosen chemical bonds of membrane lipids involved in peroxidation and the surrounding water molecules. Therefore, under the OPTIC BIOEM action a unique non-linear optical system was coupled with a very original EM exposure setup able to deliver, to biological loads in vitro, controlled E pulses covering a spread frequency spectrum in matched conditions.
Conclusion of the action: OPTIC BIOEM was a successful project since all the objectives were reached in the appropriate time. I, also, performed outstanding training and network activities during all the project period together with constant dissemination of the results as detailed in this summary. CARS hyperspectral imaging were carried out on the established samples. This type of analysis is completely new and the obtained results opened the way to a deep comprehension of electro-permeabilization mechanisms.
Thus, the use of an optical non-linear imaging technique (i.e. CARS) seems extremely suitable to follow and image, with a time resolution of few nanosecond, the dynamics of specifically chosen chemical bonds of membrane lipids involved in peroxidation and the surrounding water molecules. Therefore, under the OPTIC BIOEM action a unique non-linear optical system was coupled with a very original EM exposure setup able to deliver, to biological loads in vitro, controlled E pulses covering a spread frequency spectrum in matched conditions.
Conclusion of the action: OPTIC BIOEM was a successful project since all the objectives were reached in the appropriate time. I, also, performed outstanding training and network activities during all the project period together with constant dissemination of the results as detailed in this summary. CARS hyperspectral imaging were carried out on the established samples. This type of analysis is completely new and the obtained results opened the way to a deep comprehension of electro-permeabilization mechanisms.
Explanation of the work - Overview of progress.
WP1 We started the design looking at different planar electromagnetic structures. The chosen optimal bio-chip was fabricated during a secondment period at the “Ecole Normale Supérieure Cachan” ENS - Institut D’Alembert IDA established through a signed convention. The fabricated GCCPW were characterization trough experiments in time and frequency domains. Finally, the biochip was integrated into the CARS microscope and a suitable microscope plastic plate was fabricated to guarantee isolation of the ground of the bio-chip and the microscope.
WP2: Different types of CARS experiments were conducted. Hyperspectral images into the water vibrational band were acquired on different types of samples. We started our experiments using cells (red blood cells, DC-3F cells and haMSCs). Hyperspectral experiments were performed after exposing the cells to 8 pulses of 100 µs with variable E field amplitudes (0.16 to 0.24 MV/m) or to 100 or 200 pulses of 10 ns with an E field amplitude of 9 MV/m. Hyperspectral analysis on GUV was also carried out. Unfortunately, due to limitation in the sample concentration and the absence of the contrast, it was impossible to visualize GUV under the microscope. To overcome these difficulties alternative highly concentrated spherical synthetic systems, named liposomes, were employed. Real time acquisitions of CARS signals were also achieved. In this last protocol pulse delivery was followed by laser illumination after 1 ns implying that we were able to image chemical bounds of bio-sample under the permeabilization process.
WP3: To support CARS analysis, classical fluorescence microscopy was performed on cells (red blood cells, DC-3F, and MSC). This technique using fluorescence cell staining allows to visualize, under the microscope, the fluorescence appearance during the electric pulse application.To verify the permeabilization of GUV and liposomes, since it is not possible to observe these samples under a microscope due to the lack of contrast, fluorimetric measurements were carried out staining the sample with a suitable dye that enters into the vesicles during their formation and is then released if the vesicle membrane is permeabilized. In WP3 mass spectroscopy analyses were also achieved to detect lipid oxidation in order to support CARS data interpretation. Substantial oxidation for microsecond pulsed samples was observed while a very low oxidation level was obtained for the nanosecond exposed ones. This indication together with the difference in structuring water observed in CARS between microsecond and nanosecond pulses when applied to the bio-samples leads to hypothesize a different permeabilization dynamics in these two cases.
In WP3 we also performed microdosimetric modeling of cells in 2D and 3D.
Results exploitation:
- 7 communications to international peer reviewed conferences.
- 1 press-article.
- 1 submission to Nature Scientific Reports.
- 4 papers are under preparation.
Dissemination:
- realization of a web-page and project logo.
- organization of two dedicated seminars.
- organization of two specific workshops.
- presentation to the “Flash days of IGR”, Institute Gustave Roussy, Villejuif, France.
The training activities:
- first International Workshop on Metamaterials-by-Design December 2015.
- Institute d’Electronique Fondamentale “Nano and Microfabrication Processes and Principles”.
- COST EMF MED BM 1309 VIII Course of International School of Bioelectromagnetics “Alessandro Chiabrera”, Erice, Italy.
- “Formation for animal handling and good practices” Institute Gustave Roussy, Villejuif, France.
- habilitation to use “Plate-forme Imagerie Cellulaire et Cytométrie” of Institute Gustave Roussy, Villejuif France.
The outreach activities:
- weekly group meetings.
- LEA EBAM meeting held in Villejuif.
- ambassador of the EBTT 2016 Scientific Workshop and Postgraduate Course.
- supervision of a master thesis.
- supervision of the work of a PhD student.
- collaborations with different international groups.
- session Chair in two international conferences.
- speaker in five different internationaL conferences.
WP1 We started the design looking at different planar electromagnetic structures. The chosen optimal bio-chip was fabricated during a secondment period at the “Ecole Normale Supérieure Cachan” ENS - Institut D’Alembert IDA established through a signed convention. The fabricated GCCPW were characterization trough experiments in time and frequency domains. Finally, the biochip was integrated into the CARS microscope and a suitable microscope plastic plate was fabricated to guarantee isolation of the ground of the bio-chip and the microscope.
WP2: Different types of CARS experiments were conducted. Hyperspectral images into the water vibrational band were acquired on different types of samples. We started our experiments using cells (red blood cells, DC-3F cells and haMSCs). Hyperspectral experiments were performed after exposing the cells to 8 pulses of 100 µs with variable E field amplitudes (0.16 to 0.24 MV/m) or to 100 or 200 pulses of 10 ns with an E field amplitude of 9 MV/m. Hyperspectral analysis on GUV was also carried out. Unfortunately, due to limitation in the sample concentration and the absence of the contrast, it was impossible to visualize GUV under the microscope. To overcome these difficulties alternative highly concentrated spherical synthetic systems, named liposomes, were employed. Real time acquisitions of CARS signals were also achieved. In this last protocol pulse delivery was followed by laser illumination after 1 ns implying that we were able to image chemical bounds of bio-sample under the permeabilization process.
WP3: To support CARS analysis, classical fluorescence microscopy was performed on cells (red blood cells, DC-3F, and MSC). This technique using fluorescence cell staining allows to visualize, under the microscope, the fluorescence appearance during the electric pulse application.To verify the permeabilization of GUV and liposomes, since it is not possible to observe these samples under a microscope due to the lack of contrast, fluorimetric measurements were carried out staining the sample with a suitable dye that enters into the vesicles during their formation and is then released if the vesicle membrane is permeabilized. In WP3 mass spectroscopy analyses were also achieved to detect lipid oxidation in order to support CARS data interpretation. Substantial oxidation for microsecond pulsed samples was observed while a very low oxidation level was obtained for the nanosecond exposed ones. This indication together with the difference in structuring water observed in CARS between microsecond and nanosecond pulses when applied to the bio-samples leads to hypothesize a different permeabilization dynamics in these two cases.
In WP3 we also performed microdosimetric modeling of cells in 2D and 3D.
Results exploitation:
- 7 communications to international peer reviewed conferences.
- 1 press-article.
- 1 submission to Nature Scientific Reports.
- 4 papers are under preparation.
Dissemination:
- realization of a web-page and project logo.
- organization of two dedicated seminars.
- organization of two specific workshops.
- presentation to the “Flash days of IGR”, Institute Gustave Roussy, Villejuif, France.
The training activities:
- first International Workshop on Metamaterials-by-Design December 2015.
- Institute d’Electronique Fondamentale “Nano and Microfabrication Processes and Principles”.
- COST EMF MED BM 1309 VIII Course of International School of Bioelectromagnetics “Alessandro Chiabrera”, Erice, Italy.
- “Formation for animal handling and good practices” Institute Gustave Roussy, Villejuif, France.
- habilitation to use “Plate-forme Imagerie Cellulaire et Cytométrie” of Institute Gustave Roussy, Villejuif France.
The outreach activities:
- weekly group meetings.
- LEA EBAM meeting held in Villejuif.
- ambassador of the EBTT 2016 Scientific Workshop and Postgraduate Course.
- supervision of a master thesis.
- supervision of the work of a PhD student.
- collaborations with different international groups.
- session Chair in two international conferences.
- speaker in five different internationaL conferences.
The OPTIC BIOEM results determine a deeper comprehension of the cell permeabilization mechanism under exposure to short electric pulses useful in different and innovative therapeutic applications. I acquired a number of different skills in different disciplines which completely and complementarily contributed to form my present professional profile. The successful completion of OPTIC BIOEM provides clear evidence of my professional maturity and make me a credible candidate for positions of excellence either in research institutions, in academia or in industry, all over Europe. OPTIC BIOEM further contributed to the European excellence and to face social challenges in health and wellbeing. The excellence in science is represented by the deeper comprehension of membrane electropermeabilization mechanisms. Indeed, only in this way a real control of pulsed E field action to in vitro and subsequently in vivo models can be reached for the advancement of innovative treatments and technologies for mid-long term cancer treatments in humans. Hence, tools and technologies based on electric pulses applications for advanced therapies will potentially be more effectively developed on the basis of elementary interaction mechanism comprehension determined at the end of the OPTIC BIOEM action.