Periodic Reporting for period 1 - MecHA-Nano (Understanding cell-nanoparticle interactions through mechanobiology)
Periodo di rendicontazione: 2023-04-01 al 2025-03-31
STATE OF THE ART, PROBLEMS ADDRESSED AND PROJECT'S OBJECTIVES
The poor clinical translation of nanoparticle-based therapies over the last decade - hampered by issues such as inefficient targeting and therapy delivery – calls for deeper understanding of the biology of cell-nanoparticle interactions. In this context, targeting mechanosensing-activated cell pathways can emerge as a viable and promising option to guide cell fate and readdress cell functions. Mechanosensing is the way by which cells perceive and convert extracellular mechanical stimuli, such as cell-cell contact and fluid pressure, into intracellular biochemical signals to adapt their behavior to the environment. Importantly, mechanosensing components can control the expression of genes involved in the cell’s migration, survival and resistance to drugs, through the recruitment of transcriptional factors, which are proteins or group of proteins that can bind specific region of DNA and promote the translation of the associated gene. Nowadays, it is clear how the different proteins of the mechanobiology pathways affect the adhesion, spreading and differentiation of cells growth on different substrates. The deletion of relevant components of the mechanosensing machinery, such as Yes-associated Protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), alter cells’ behaviour and stiffness. This has important consequences on the expression of genes and proteins, and ultimately on the cell functionalities. However, while the study of Mechanobiology-on-a-chip may provide important information about the behavior of cells, the use of colloidal suspensions of nanoparticles (NPs) is more desirable for broader approaches. So far, the mechanobiological effects of colloidal nanoparticles on cells were not investigated. Nevertheless, the possibility to tune the cell’s mechanosensing may disclose important aspects of nanomaterials’ design, widening their applications and revolutionizing the nanomedicine field. This project aims to address the response of Hippo pathway on cells upon interaction with NPs. The first objective will be the synthesis of nanoparticles of different size, shape and stiffness, using a silica scaffold coated with hyaluronic acid. The second objective will consist in the application of Super-Resolution Microscopy (STORM and ExM) for studying cell-nanoparticle interactions with unprecedented details and unveil the interaction/structure/spatiotemporal localization of mechanosensing components related to the Hippo pathway (i.e. YAP, actin and focal adhesions) at molecular level. The third objective will be the deep analysis of the molecular biology and biochemistry of mechanosensing proteins (i.e. YAP and TAZ), and their downstream effectors (i.e. TEAD, transcriptional factors) involved in the response to NP-cell interaction. The forth objective will pursue the analysis of cell-nanoparticle interactions using NenoVision technology (LiteScopeTM), for measuring cell stiffness at the boundary of NP-cell contact with unique resolution.
RELEVANCY FOR THE SOCIETY
The outcomes of this research project may be potentially useful for several applications and may attract wide interest of entities working in the healthcare system. By revealing how particles affect the mechanobiology of healthy cells and damaged/malignant cells, it become feasible to develop a new class of devices for biomedical purposes. Some applications that can benefit from the discoveries of this project can be easily found: (i) it would be possible to sort out malignant cells from a bulk cell sample, based on the different cells’ responses upon particles treatment; (ii) sensors can be designed, to better study the behavior of unhealthy cells in different diseases and then determine the best therapeutic option for a specific patient (personalized medicine); (iii) it can make feasible to control the organization of organoids for studying complex biological systems in vitro or control the differentiation/regeneration of tissues in vivo.
The poor clinical translation of nanoparticle-based therapies over the last decade - hampered by issues such as inefficient targeting and therapy delivery – calls for deeper understanding of the biology of cell-nanoparticle interactions. In this context, targeting mechanosensing-activated cell pathways can emerge as a viable and promising option to guide cell fate and readdress cell functions. Mechanosensing is the way by which cells perceive and convert extracellular mechanical stimuli, such as cell-cell contact and fluid pressure, into intracellular biochemical signals to adapt their behavior to the environment. Importantly, mechanosensing components can control the expression of genes involved in the cell’s migration, survival and resistance to drugs, through the recruitment of transcriptional factors, which are proteins or group of proteins that can bind specific region of DNA and promote the translation of the associated gene. Nowadays, it is clear how the different proteins of the mechanobiology pathways affect the adhesion, spreading and differentiation of cells growth on different substrates. The deletion of relevant components of the mechanosensing machinery, such as Yes-associated Protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), alter cells’ behaviour and stiffness. This has important consequences on the expression of genes and proteins, and ultimately on the cell functionalities. However, while the study of Mechanobiology-on-a-chip may provide important information about the behavior of cells, the use of colloidal suspensions of nanoparticles (NPs) is more desirable for broader approaches. So far, the mechanobiological effects of colloidal nanoparticles on cells were not investigated. Nevertheless, the possibility to tune the cell’s mechanosensing may disclose important aspects of nanomaterials’ design, widening their applications and revolutionizing the nanomedicine field. This project aims to address the response of Hippo pathway on cells upon interaction with NPs. The first objective will be the synthesis of nanoparticles of different size, shape and stiffness, using a silica scaffold coated with hyaluronic acid. The second objective will consist in the application of Super-Resolution Microscopy (STORM and ExM) for studying cell-nanoparticle interactions with unprecedented details and unveil the interaction/structure/spatiotemporal localization of mechanosensing components related to the Hippo pathway (i.e. YAP, actin and focal adhesions) at molecular level. The third objective will be the deep analysis of the molecular biology and biochemistry of mechanosensing proteins (i.e. YAP and TAZ), and their downstream effectors (i.e. TEAD, transcriptional factors) involved in the response to NP-cell interaction. The forth objective will pursue the analysis of cell-nanoparticle interactions using NenoVision technology (LiteScopeTM), for measuring cell stiffness at the boundary of NP-cell contact with unique resolution.
RELEVANCY FOR THE SOCIETY
The outcomes of this research project may be potentially useful for several applications and may attract wide interest of entities working in the healthcare system. By revealing how particles affect the mechanobiology of healthy cells and damaged/malignant cells, it become feasible to develop a new class of devices for biomedical purposes. Some applications that can benefit from the discoveries of this project can be easily found: (i) it would be possible to sort out malignant cells from a bulk cell sample, based on the different cells’ responses upon particles treatment; (ii) sensors can be designed, to better study the behavior of unhealthy cells in different diseases and then determine the best therapeutic option for a specific patient (personalized medicine); (iii) it can make feasible to control the organization of organoids for studying complex biological systems in vitro or control the differentiation/regeneration of tissues in vivo.
At the early stage of the project, the definition and optimization of the parameters for the reproducible synthesis of nanoparticles coated with functional biological moieties, i.e. hyaluronic acid, with different size, shape and stiffness will be achieved. According to the synthesis reported by Caruso F. and co-workers at the partner institution (The University of Melbourne), rod-shape, cubic-shape and spherical-shape nanoparticle templates will be grown. By varying the concentration and the reaction time, particles with different aspect ratio will be obtained. Once nanoparticle with the desired properties have been obtained, in vitro test of the basic bio-nano interactions (i.e. toxicity and internalization pathways) on breast cancer cell lines will be conducted.
A wide collection of different nanoparticles, suitable for creating a Bio-Nano interactions data bank, will be generated. The broad amount of data will provide an unprecedented guide for the future development of mechanotargeting research.
Use of advanced super-resolution microscopy techniques, such as ExM and STORM, for the in depth study of the interactions between particles and living systems, will be implemented along the project and will provide unprecedented resolution of cell-nanoparticle interactions.
The application of nanotechnology on the mechanobiology response induced on the cells by colloidal nanoparticles is one of the innovative aspects of the MecHA-Nano project. By using nanoparticles with different size, shape and composition allows to understand an extremely broad range of cells response to nanomaterials’ treatment.
The exploitation of innovative cell culture techniques, from bioreactor setups to organoids, to asses cell-nanoparticle interactions will allow to conduct the experiments in physiological-like conditions for better designing nanomaterials and determining their response in living organisms, to re-calibrate the in vivo studies’ needs and support 3Rs policy.
Thanks to the collaboration with NenoVision company, the implementation of LiteScope (TM), provided with an AFM-SEM mode never used before for studying cell-nanoparticles interaction, will generate unprecedented precision and resolution for addressing the questions arisen by the cell-nanoparticle contact (stiffness, morphology and topography)
Use of advanced super-resolution microscopy techniques, such as ExM and STORM, for the in depth study of the interactions between particles and living systems, will be implemented along the project and will provide unprecedented resolution of cell-nanoparticle interactions.
The application of nanotechnology on the mechanobiology response induced on the cells by colloidal nanoparticles is one of the innovative aspects of the MecHA-Nano project. By using nanoparticles with different size, shape and composition allows to understand an extremely broad range of cells response to nanomaterials’ treatment.
The exploitation of innovative cell culture techniques, from bioreactor setups to organoids, to asses cell-nanoparticle interactions will allow to conduct the experiments in physiological-like conditions for better designing nanomaterials and determining their response in living organisms, to re-calibrate the in vivo studies’ needs and support 3Rs policy.
Thanks to the collaboration with NenoVision company, the implementation of LiteScope (TM), provided with an AFM-SEM mode never used before for studying cell-nanoparticles interaction, will generate unprecedented precision and resolution for addressing the questions arisen by the cell-nanoparticle contact (stiffness, morphology and topography)