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“Tracking nano-bioprocesses using Super-Resolution Microscopy Techniques”

Periodic Reporting for period 2 - NANO-SUPREMI (“Tracking nano-bioprocesses using Super-Resolution Microscopy Techniques”)

Reporting period: 2018-03-01 to 2020-02-29

Research objective
Nano-Supremi aims to uncover and exploit novel physical, chemical and biological properties of bio-nanodevices that can be utilised at the micro- and macro-scale for the mastering of intelligent materials and new diagnostic tools.

Zooming on intracellular trafficking of nanomedicines and biomolecules: We are applying super-resolution and advanced microscopy techniques (SIM, STORM, STED, FLIM, FCS) to quantitatively and reliably assess the size, shape, intracellular trafficking, targeting and efficacy of newly developed nanomedicines and DNA molecular switches. This will ultimately result in the evaluation of in vitro protein activity and therapeutic efficacy of nanomedicines, filling the gap between innovative research and its exploitation in clinical setting.
Three case studies were set up to investigate nanoscale synthetic and biological structures and to probe nanoscale dynamic processes.

1- Intracellular targeting and monitoring of Transcription Factor proteins (NF-kB and TEAD) in living cells.
2- Intracellular and quantitative detection of shuttling proteins (YAP/TAZ) in living cells.
3- Intracellular trafficking of siRNA based nanomedicines to downregulate YAP protein.

Training objective:
We are providing young scientists, post-doctoral researchers and academic staff, with cross-disciplinary training in molecular biology, optics, bio-imaging, nano-biomaterials, bioanalytical chemistry. Nano-Supremi programme promotes a transfer of knowledge by experimental training, joint workshops and seminars, between research and educational institutions located in Italy, Australia, Czech Republic, Unites States, Cuba and Brazil.

The challenges for personalizing healthcare represent an important priority within European policies. In particular, RNA and DNA-based therapeutics have rapidly advanced from research discovery to preclinical and clinical trials. One of the primary challenges associated with cancer therapy is delivering the therapeutic agent to the tumour in a safe and effective manner. Given their peculiar behaviour and function within the cell, it is overseen that the targets protein chosen (Nf-kB, NFAT, YAP/TEAD) in this project will be used as diagnostic markers for pathologies like cancer and cardiovascular diseases.
Nano-Supremi is developing bio-imaging methods to non-invasively visualise biological processes, triggered by nanomaterials, in real time, for therapy (cancer therapy, regenerative medicine) and diagnostics.
The development of innovative therapeutic and diagnostic products and technologies could boost growth, and the economic impact of biotechnology, pharmaceutical, optics companies, especially SMEs, dealing with the manufacturing of drug-delivery platforms. This is a niche where Europe can gain prestige and a corresponding share of the global market for bio-nanomaterials.
We applied super-resolution and advanced microscopy techniques (SIM, STORM, STED, FLIM, FCS) to quantitatively and reliably assess the size, shape, intracellular trafficking, targeting and efficacy of newly developed nanomedicines and DNA molecular switches. In particular, we discovered that the DNA nanoswitch serves as a dynamic probe for sensing and comparatively quantifying in real time the intracellular silencing of NF-B, with no need for reporter genes. This TF-responsive nanoswitch can be potentially used as a simple, rapid, functional sensing device as an alternative to standard antibody-based staining techniques (based on a static structural matching in fixed cells) and multi-step and reagent-intensive reporter gene technologies. These important results have been published on Nanoscale 2018, DOI: 10.1039/C7NR07814E
Progress beyond the state of art:
The true impacts and capabilities of fluorescence-based super-resolution microscopy are still emerging. The visualization and time-resolved monitoring of the interactions of nanomaterials with biological entities at high resolution in 3D is a powerful tool. The internalization or association of nanomaterials with cells and tissues is of relevance for nearly any biologically oriented processes. In first two years of implementation of Nano-Supremi, we addressed the following question:
Can we track nanometer-scale bioprocesses such as cellular uptake, intracellular trafficking and targeting of nanomedicines (DNA, siRNA and microRNA) and biosensors using advanced microscopy techniques?

To achieve these goals a great effort in terms of interdisciplinary collaboration between centres of excellence, sharing highly specialized expertise in optics/bioimaging, materials science chemistry, biochemistry and biosensing and cell biology, was required.

Expected results

Socio-economic implications of the project
Nano-Supremi is training a new generation of nanotechnologists, biotechnologist and micro-nanoscopists. Nano-Supremi is giving European and Intenational researchers from United States, Australia, Brazil and Cuba, the opportunity to undertake highly innovative and potentially transformational research. An ambitious goal of Nanosupremi is bringing together people, ideas, and solutions from across different disciplines, sectors, ideologies, and traditions. We utilized advanced scientific knowledge and infrastructures as a tool of diplomacy to mitigate cultural differences and establish a common ground across boundaries powering 21st century innovation.

Communication and leadership skills: During the secondments, researchers developed communication skills by attending regular research group meetings, departmental seminars, and scientific workshops. The researchers joined the tutoring activities and demonstration courses. ESR and ER developed leadership skills by applying for joint research grants.
Developing new and lasting research collaborations at the European and global levels:
This project leveraged existing productive research collaborations, and reciprocal access for European scientists and early-stage researchers to facilities and expertise not readily available in Europe. Nano-Supremi is giving European researchers the opportunity to build new knowledge in international laboratories that carry out cutting-edge research.
Nano-Supremi allows the consortium to continue and strengthen the ongoing scientific collaborations and to further enlarge the collaborative network, making Europe attractive to researchers from all over the world.

Giving the young a taste for research and careers in science:
By building an international environment and a dynamic work-place with mobility opportunities Nanosupremi encouraged early-stage researchers to start a career in science in the European laboratories involved in this project. Three early-stage researcher involved in Nanosupremi have been awarded MSCA-IF-GF Global Fellowships.

Greater place and role for women in research: We integrated a gender dimension in Nano-Supremi research, innovation and management. The coordinator of the project is a mid- career female scientist 44% of the exchanged researchers were female scientists.