Periodic Reporting for period 1 - MAGPIEZ (Tunning the force for remote magnetomechanical gating of Piezo1 channels)
Reporting period: 2022-07-01 to 2024-06-30
In a process called mechanotransduction, our cells use a set of receptors capable of sensing mechanical forces from their environment and convert them into electrochemical signals, triggering cellular responses. Among these receptors, the Piezo1 protein has emerged as a crucial mechanosensitive (MS) ion channel, highly implied in various normal and pathological processes. For instance, cardiovascular systems are one of the most relevant targets to study mechanotransduction linked to Piezo1 due to the diversity of aspects in which it is involved, including development stages, physiology, and pathologies, such as atherosclerosis, control of blood pressure, and ischemic injury. Therefore, they hold great promise as potential novel therapeutic target. However, and despite their great importance, the full mechanism of how Piezo responds to a force and transduces it into pore opening remains largely unknown. Therefore, the development of new tools for the study and remote control of these MS channels is a crucial currently challenge.
The MAGPIEZ project aims at developing and validating a novel platform that uses small MNPs to study mechanotransduction linked to Piezo1 channels in endothelial cells through remote magnetic stimulation obtaining real-time responses. The developed multifunctional nanoplatform is: i) specific: MNPs bind to Piezo1 selectively; ii) non-invasive: no need for prior cell modification; iii) precise: small MNPs to gain control at the molecular and sub-molecular level; iv) with remote and fast spatiotemporal response: digitized output signals in response to magnetic input cues and v) real time monitoring: magnetic applicators integrated in a fluorescence microscope.
These goals will be accomplished by three specific objectives: 1) To develop a toolkit including, i) a surface engineered MNPs with tuneable magnetism to exert high mechanical forces and able to selective target endogenous human Piezo1 channel and ii) a dedicated magnetic applicator able to deliver diverse magnetic cues. 2) To investigate the possibility to open Piezo1 upon magnetic switching, and to activate important intracellular pathways connected with increase of calcium influx inside endothelial cells, without prior cellular modification; 3) Validation of the magnetomechanical activation of Piezo1 channels in a more realistic environment, performing the experiments in a presence of fluid pressure recreating the vascular network.
The MAGPIEZ project aims at developing and validating a novel platform that uses small MNPs to study mechanotransduction linked to Piezo1 channels in endothelial cells through remote magnetic stimulation obtaining real-time responses. The developed multifunctional nanoplatform is: i) specific: MNPs bind to Piezo1 selectively; ii) non-invasive: no need for prior cell modification; iii) precise: small MNPs to gain control at the molecular and sub-molecular level; iv) with remote and fast spatiotemporal response: digitized output signals in response to magnetic input cues and v) real time monitoring: magnetic applicators integrated in a fluorescence microscope.
These goals will be accomplished by three specific objectives: 1) To develop a toolkit including, i) a surface engineered MNPs with tuneable magnetism to exert high mechanical forces and able to selective target endogenous human Piezo1 channel and ii) a dedicated magnetic applicator able to deliver diverse magnetic cues. 2) To investigate the possibility to open Piezo1 upon magnetic switching, and to activate important intracellular pathways connected with increase of calcium influx inside endothelial cells, without prior cellular modification; 3) Validation of the magnetomechanical activation of Piezo1 channels in a more realistic environment, performing the experiments in a presence of fluid pressure recreating the vascular network.
During the MAGPIEZ project, we performed several scientific and technical activities directly linked to the objectives of the project, including:
1. Development of MAGPIEZ toolkits.
-Several magnetic nanoparticles (MNPs) based on manganese-iron oxide MNPs and mixed ferrites (zinc-manganese-iron oxide MNPs) were obtained by the one-step thermal decomposition method. We tuned critical experimental parameters to obtain different sizes (between 13 - 26 nm), shapes (cubes, octahedrons, disks), and compositions.
-Design and characterization of a versatile magnetic applicator able to exert mechanical forces using a slowly rotating direct current magnetic field. Theoretical calculations of pulling and/or torque forces generated by MNPs depending on magnetic strength and distances.
-Selectively and directly target endogenous Piezo1 channels by oriented conjugation on MNPs surface of an antibody against Piezo1.
2. Remote magneto-mechanical activation of Piezo1 channel in the endothelial cell line. The gate of the Piezo1 channel by optimized MNPs was followed by calcium increase influx using time-lapse fluorescence microscopy.
3. Study the downstream pathways activations due to Piezo1 gating and the sustained calcium increase inside cells.
4. Validation of the potential of MAGPIEZ toolkits to stimulate signalling pathways related to cell proliferation in a 3D vascular network developed using a microfluidic chamber. We evaluated the impact of shear stress forces at physiological level (≤5 dyne/cm2) on calcium increase in HUVECs by applying shear stress in combination with MAGPIEZ nanoplatform
1. Development of MAGPIEZ toolkits.
-Several magnetic nanoparticles (MNPs) based on manganese-iron oxide MNPs and mixed ferrites (zinc-manganese-iron oxide MNPs) were obtained by the one-step thermal decomposition method. We tuned critical experimental parameters to obtain different sizes (between 13 - 26 nm), shapes (cubes, octahedrons, disks), and compositions.
-Design and characterization of a versatile magnetic applicator able to exert mechanical forces using a slowly rotating direct current magnetic field. Theoretical calculations of pulling and/or torque forces generated by MNPs depending on magnetic strength and distances.
-Selectively and directly target endogenous Piezo1 channels by oriented conjugation on MNPs surface of an antibody against Piezo1.
2. Remote magneto-mechanical activation of Piezo1 channel in the endothelial cell line. The gate of the Piezo1 channel by optimized MNPs was followed by calcium increase influx using time-lapse fluorescence microscopy.
3. Study the downstream pathways activations due to Piezo1 gating and the sustained calcium increase inside cells.
4. Validation of the potential of MAGPIEZ toolkits to stimulate signalling pathways related to cell proliferation in a 3D vascular network developed using a microfluidic chamber. We evaluated the impact of shear stress forces at physiological level (≤5 dyne/cm2) on calcium increase in HUVECs by applying shear stress in combination with MAGPIEZ nanoplatform
The most important achievement of the MAGPIEZ project is that we have developed a novel, non-invasive tool for the remote gating of endogenous Piezo1 channels in situ using small MNPs. We have addressed a crucial challenge, as up to date all studies were performed using cells over expressing Piezo1 channels. This can help to take the first steps toward improving magnetogenetic tools for clinical translation.