Periodic Reporting for period 2 - NanoTBTech (Nanoparticles-based 2D thermal bioimaging technologies) Reporting period: 2019-09-01 to 2022-02-28 Summary of the context and overall objectives of the project The Nanotech's goal was to develop a new 2D time-resolved temperature readout, overcoming the conundrum represented by those biomedical imaging demands that cannot be handled with any thermometric method currently available.The overall objectives were:1) Design and fabrication of near-infrared(NIR) emitting nanoparticles and heater-thermometer nanostructures.2) Understanding the thermographic parameters of the nanothermometers and heater-thermometer nanostructures.3) Optimization and in vitro/in vivo incorporation of luminescent, nontoxic, long-term biodegradable, long-circulating, tumour-targeted and heating/sensing nanoplatforms.4) Developing new technology for in vitro/in vivo (small animal level) simultaneous luminescent 2D thermal imaging and optical microscopy imaging for localized controlled hyperthermia in cancer cells and tumour microenvironment.5) Explore in vivo time-gated and 2D magnetic-or optical-gated thermal transient thermometry in murine models of liver metastasis.While proof-of-concept thermal sensing using luminescent NPs has been already demonstrated by numerous groups worldwide (including NanoTBTech consortium members), remote 2D thermal imaging was (in 2019, when the project started) a tremendous challenge implying: i)the fabrication of novel nanostructures with substantially improved thermal sensitivities in first (BW-I,650-1000nm) second (BW-II,1000–1350nm) and third (BW-III,1550–1870nm) biological windows, and embedding novel optical functionalities with tumour targeting properties and long-term biodegradation; and ii)the development of technological solutions and dedicated instruments, yet not commercially available.To achieve these ambitious breakthroughs, nontoxic NIR emitting nanostructures, operating essentially beyond 1000 nm (where tissues become partially transparent) and combining nanothermometry and nanoheating were implemented and tested in vitro and in vivo. To monitor the emission thermal dependence (triggered by magnetic or optical heating sources) a dedicated new imaging platform was developed leading to major advances in 2D thermal bioimaging technologies: in vitro and in vivo magnetic/optical hyperthermia and in vivo time-resolved thermal images for detection and dynamical monitoring of tumours. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The overall objectives of the NanoTBTech project are fully attained and the work carried out included: i)the design and fabrication of several luminescent nanothermometers and heater-thermometer nanostructures and the selection of the two most promising nanomaterials for in-vitro and in-vivo imaging (Eu3+/Sm3+-doped polymer-based NPs and Ag2S NPs); ii)its structural, morphological, magnetic, photothermal, optical and thermometric/thermographic characterization; iii)optimised experimental setup for light-to-heat conversion efficiency determination; iv)the use of models to validate and predict luminescent thermometers (both single ion and energy-transfer based examples); v) the functionalization/PEGylation of the NPs; vi)the biosafety, cytotoxicity, and biocompatibility evaluation (in different cell lines) of some of the particles and nanostructures developed in the project; and the implementation in vitro and in vivo (small animal level) of luminescent 2D thermal imaging and optical microscopy imaging for tumour detection and localized controlled hyperthermia in cancer cells and tumour microenvironment.The project activities and results have been disseminated thanks to a website and logo, flyers, newsletters, participation in conferences, general public events and social media. The project led so far to 85 scientific publications in high-quality journals. These papers were published in collaboration with colleagues from institutions all over the world (more than 25 countries) and the fruitful cooperation between all the NanoTBTech partners is also illustrated in the number of co-authored publications (ca. 20%). Among the published papers, a handful of review and perspective articles about standardization in metrology and measurements were published, contributing to consolidating the research field. Besides several consortium meetings, we organized a winter school for PhD students and researchers on Luminescence Thermometry, 2 cycles of Webinars and, an Industrial Workshop.The potential IP and the exploitation of the results resulted in 3 national (Spain,Portugal,Poland) and 2 international (USA,Brazil) patents. Potentially exploitable results are i)a new technology joining 3D imaging in the NIR spectral region and temperature mapping using the PhotonIMAGER Short Wave Infra-Red(SWIR) in vivo temperature-imager prototype of BiospaceLAB; and ii)the use of Ln3+-doped micelles as a new class of cell stain dye that can do the ordinary organelle visualization functions of typical organic dyes (eg DAPI), and at the same time yields the absolute temperature of the targeted organelle. 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) NanoTBTech was an ambitious high risk/high gain project to supply a decisive breakthrough in non-invasive 2D luminescent thermal monitoring and imaging technology–which proof of concept was illustrated and demonstrated in two biomedical challenges:magnetically-or optically-induced local controlled hyperthermia and non-invasive, fast and deep-tissue tumour detection.NanoTBTech has succeeded in changing the landscape of real-time non-invasive imaging, providing a new kind of technology that supplied game-changing insights over physiological processes, perturbations or ongoing therapies, all of them featuring the common aspect of a biomedically-relevant thermal load to assess. The innovations developed in the frame of NanoTBtech enabled the development of a dedicated imaging platform with unprecedented performance, leading to the following advances in 2D thermal imaging technologies:•Intracellular temperature imaging, including a new class of cell stain dye (Ln3+-based polymeric micelles) for visualization and thermometry;•Real-time monitorization of intracellular magnetic hyperthermia;•Assessment of the hypothesis of “local magnetic hyperthermia” as a non-invasive cancer treatment;•Software capable of real-time analysis of fluorescence images for deep tissue/whole-body imaging;•Integration of a high-frequency magnetic field source into a small animal imaging system for imaging and subcutaneous thermal control during magnetic hyperthermia;•Optimization of the optical set-up of the new imaging prototype developed by NanoTBTech for providing spectral sensitivity;•Studies on the thermal signatures caused by tumour development.These results caused novel insights into cell pathology and physiology, heat transfer at the nanoscale, and non-invasive detection of subcutaneous anomalies, in turn, contributing to the development of novel theranostic tools. Physicians at the pre-clinical and clinical levels will be freed of the inaccurate temperature readouts (typical from mid-infrared thermal cameras, currently used to measure temperatures, see) and the step forward that NanoTBTech proposed makes feasible to know the inner temperature of the region of interest, not just the surface temperature. That represents a deep transformation of the biomedical landscape, creating a new branch of sensing technology, so-called luminescence thermal imaging. Luminescence thermometry is a very active research area and presents a particular opportunity for Europe to maintain a competitive advantage, and NanoTBTech has contributed to this effort. figure-to-be-attached-to-the-project-small.png
