FIDELGLIO will endeavor to understand the mechanisms of PEF effects on brain vasculature and immune responses in the glioblastoma microenvironment. It will employ multiphoton microscopy to reveal the mechanisms of PEFs-induced BBB disruption in vivo and apply this novel knowledge to develop a PEFs-based platform for brain treatments. It will also investigate whether PEF delivery causes an immunogenic effect that can be leveraged with other treatments (ie, targeted immunotherapy).
The objectives of FIDELGLIO are to :<br />• To engineer a flexible organic electronic microelectrode implant for delivery of pulsed electric fields in a mouse glioblastoma model<br />• To demonstrate the capability of the implant to deliver PEFs in vivo in mouse experiments while assessing the influence on vasculature perfusion and BBB disruption with multiphoton microscopy<br />• To evaluate the therapeutic impact of PEFs on glioblastoma tumors in vivo<br />• To study the influence of PEFs on the immune system in the tumor microenvironment<br />• To pave the way to a clinical application of this new type of treatment
Fabrication of the first prototype
The electrodes will be fabricated using an adapted photolithographic technique suitable for organic layers, as described in . In short, a parylene sacrificial layer is deposited (Specialty Coating Systems) on a glass substrate and patterned using photolithography, to form a contact mask on which the conducting polymer is deposited. Mechanical lift-off of the parylene leaves behind a patterned conducting polymer film. Metal (such as Pt or Au) contact pads will be pre-patterned on the substrate using lithography to facilitate reproducible electrical contact with the probes of a probe station. The sterilization of devices before integration with the biological tissue will be accomplished simply by dipping in 70% ethanol and drying in the laminar flow hood (Thermo).
The electrical impedance and electrochemical behavior of electrodes will be characterized by fast cyclic voltammetry (CV) (Autolab Potentiostat/Galvanostat). The CV behavior and charge injection limits of PEDOT:PSS electrodes will be characterized in vitro.
Measurement of PEF influence on vasculature and BBB in vivo
In this work we will be examining the PEF-induced vascular changes, which may include events such as vasodilatation, constriction and permeability leading to extravasation. These events will be observed intravitally through the cranial window and the transparent OED device using in vivo fluorescence multiphoton microscopy. A combination of macrofluorescence and multiphoton microscopy will be carried out to study these electric field responses as we attempt to permeabilize the BBB in vivo.
We were able to find the optimal thickness of PEDOT :PSS coating to maximally deliver charge to biological tissue with stability. First in vivo experiments carried out in Hungary by MINES postdoc Attila Kaszas.
The first electrode design had interdigitated fingers distanced too close, which interefered with the 2photon imaging process and limited biological field of view during imaging in addition to causing shadows Some electrode damages were observed when these were exposed to the laser intensity required for fluorescence excitation in the AMU-NeuroInflam mouse model. F. Debarbieux (AMU) suggested new specifications to circumvent this drawback while maximizing the field of view. New electrodes were then quickly redesigned.
Vessel permeability experiments were completed to schedule and we established the threshold for electrical stimulation for blood-brain-barrier disruption and vascular effects.
At CERIMED/AMU, the new implantation protocol proved long-term stability and long term clarity over 2 months in heatlhy control animals despite the lack of direct contact between the glass and dura mater normally required to avoid fibrotic tissue development.
Laurie Ladame, a young engineer has been hired at AMU and trained to perform qualitative surgeries, intravital 2P imaging, and implantable glioblastoma spheroids cultures. Whereas training to implant spheroid had to be post-poned due to COVID conditions, efforts were instead focussed on the optimization of the image analysis pipeline with the Arivis commercial software.
Initial experiments in the first few transgenic AMU-Neuroinflam mice indicated that the PEDOT:PSS electrodes did not cause reactive inflammatory responses, but that electrical stimulation did cause activation of EYFP+ microglia if not the entire innate immune system.
A stimulator unit from MINES was transfered and set-up in AMU for evaluating the impact of electrical stimulation on cerebral inflammation.
-Success of first implantation in mice and bi-photon microscopy imaging through transparent organic flexible electrodes
-Validation of biocompatibility, immune response and vascular effect triggered by electrical stimulation.
International Revues à comité de lecture 1. Dijk, Ruigrok, O’Connor. Influence of PEDOT:PSS Coating Thickness on the Performance of Stimulation Electrodes. Advanced Materials Interfaces. 7(16), 2020
Ouvrages ou chapitres d’ouvrage 1. 2.
Communications (conférence) 1. Ruigrok, Dijk, O’Connor Electropulsation in glioblastoma cells by plastic microelectrode devices designed for live cell fluorescence imaging. BioEM Portoroz June 2019
France Revues à comité de lecture 1. 2.
Ouvrages ou chapitres d’ouvrage 1. 2.
Communications (conférence) 1. Ruigrok, Dijk, O’Connor Electropulsation in glioblastoma cells by plastic microelectrode devices. ISEBTT Toulouse Sept 2019
2. Dijk G, Ruigrok H, O’Connor R. Characterization of the Conductive Polymer PEDOT:PSS as a Material for Electrical Stimulation ISEBTT Toulouse Sept 2019
3. Lefevre M, O’Connor D, Donahue MJ, Bardet SM, O’Connor RP. Development of flexible organic microelectrodes for the detection and the treatment of brain cancer. ISEBTT Toulouse Sept 2019
Actions de diffusion Articles de vulgarisation
Conférences de vulgarisation 1. 2.
Liste des publications monopartenaires (impliquant un seul partenaire)
International Revues à comité de lecture 1. 2.
Ouvrages ou chapitres d’ouvrage 1. 2.
Communications (conférence) 1. Invited session: O’Connor RP. In vivo flexible, implantable, bioelectronic devices for preclinical investigations. World Congress on Electroporation, ISEBTT Toulouse Sept 019
FIDELGLIO will develop and test a flexible implantable organic electronic device for the treatment of glioblastoma (GBM) with pulsed
electric fields. We will use an intravital multiphoton imaging approach to evaluate efficacy at the cellular scale, both in the tumor
and its environment, in a syngenic, orthotopic murine model of GBM. We will determine the threshold of pulsed electric fields
required to transiently disrupt the blood brain barrier, with the view of improving drug delivery and immune system access to the
tumor. FIDELGLIO will quantify the direct influence of pulsed electric fields on tumor cell proliferation and death, angiogenesis, and
the innate immune response against GBM. With the established treatment protocol, we will demonstrate the therapeutic relevance
of our approach to eradicate cancer cells remaining at the margin of the resected tumor. The project has the potential to deliver a
novel, safe treatment for GBM that can rapidly reach the medical market for humans.
Rodney O'Connor (Ecole Nationale Superieur de Saint-Etienne -Institut Mines Telecom)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
EMSE-CMP Ecole Nationale Superieur de Saint-Etienne -Institut Mines Telecom
AMU_INT Aix-Marseille Université_Institut de Neurosciences de la Timone
Help of the ANR 575,438 euros
Beginning and duration of the scientific project: January 2019 - 36 Months