Periodic Reporting for period 2 - CARBO-IMmap (Immune activity Mapping of Carbon Nanomaterials) Reporting period: 2019-03-01 to 2022-02-28 Summary of the context and overall objectives of the project Long-term nanobiotechnology goal: The long-range goal of Carbo-IMmap is to develop a functional pipeline for the immune-characterization of carbon nanomaterials, for the qualitative and quantitative assessment in vitro and ex vivo of the human immune compatibility and immune activity of newly developed carbon materials.Long-term nanomedical goal: The functional pipeline will be used for the accurate immune mapping of graphene oxide, aminated graphene, exfoliated graphene, graphene nanoribbons, functionalized carbon nanotubes and carbon nanotube fibers. The accurate immuno-characterization of the tested materials on the basis of intrinsic physical-chemical features and immunological properties will open breakthrough perspectives for the development of new therapeutic approaches applying nanomaterials as immunomodulators, immunotherapetutic agents, molecular vectors and scaffolds for tissue regeneration/engineering.Key implementation aspects: The innovative pipeline will ground on computational modelling systems to predict the materials behaviours in the biological fluids and their interaction with biomolecules. The modelling strategy will be coupled with a wide array of high throughput assays based on immune-phenotypic and immune-genomics analysis providing the simultaneous detection of multiple effects on the five major immune cell populations and ex vivo on the highly complex immune blood cell pool. The combination of the most innovative technologies (12-colour Flow cytometry, ImageStream, CyTOF, single cell gene expression), allowing multiplex analysis of many cell types, will provide a forefront approach to investigate the immune impact.Scientific impact: The successful development and optimization of the functional pipeline for the characterization of the immune activity of newly synthesized, stable carbon nanomaterials will greatly advance the engineering process of carbon nanomaterials. The implication of this project extend beyond the specific nanoscience program. The unprecedented testing of 5 different types of materials to assess jointly the effect of different parameters (such as size and chemical functionalization) on all major type of immune blood cells, is a step ahead in basic research attempting to predict nanomaterial immune activity and to take advantage of their potential immunomodulation feature. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the frame of this project, various carbon nanomaterials, spanning from graphene oxide to nanodiamonds have been prepared and/or functionalized and their immune characterizations have been deeply studied taking into account the effect of different appendages and also of the effect of size can have on the immune response. It was also possible to analyze the genomic response and the complex effect on the activation or suppression of gene expression.These results have been reported in more the 30 papers and in a number of communications to symposia, meeting and congresses. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The main project innovation is an outstanding paradigm shift in the nanomaterial engineering and classification. The inclusion of the analysis of the immunological effects when designing new nanomaterials, for biomedical applications, aims at turning them from simple carriers/scaffold into active molecules endowed with immune modulatory properties, which can be tailored adjusting their physicochemical properties. Our approach will be of advantage for the engineering of highly specific nanomaterials in different immune response contexts. For example, the availability of vectors endowed with intrinsic properties which can either activate the antitumor immunity or by-pass the immune suppression mechanisms, put in place by tumour cells, could provide a step forward in cancer therapy, finding applications as cancer vaccines or tumor immunotolerance suppressors. Potentialities of CNSs' response
