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Thermometry and Photoacoustic-Imaging Outstanding Nanoprobes

Periodic Reporting for period 1 - TEMPTATION (Thermometry and Photoacoustic-Imaging Outstanding Nanoprobes)

Reporting period: 2017-04-01 to 2018-11-30

Nowadays, medical imaging has become a widely needed tool to effectively detect and fight a gamut of diseases, thus becoming a paramount factor to promote higher life expectancy and well-being worldwide. Optical imaging, and more specifically fluorescence imaging (hereafter FI), is a “younger relative” among the family of medical imaging modalities such as magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT; based on X-rays) and ultrasound (US).

Dynamic (patho-) physiological processes can be feasibly tracked in real time by means of FI, unveiling key metabolic aspects still unknown. Second, and much closer to the clinical level, both such signal collection speed plus the absence of post-processing requirements after image acquisition make of FI the ideal intra-operative guide for surgical resection of tumor margins. Although modern surgical advancements have improved surgical oncology, adequate tumor visualization remains a limitation preventing total removal. Surgeons rely primarily on white light reflectance, which limits the differentiation between healthy issue and tumor and can lead to residual cancer cells inadvertently left behind at the resection border. FI also has the potential to distinguish different anatomic structures to reduce inadvertent injury to healthy tissue. For none of these two relevant applications above exists an actual alternative to FI.

Core-ingredient for credible fluorescence in vivo imaging: picking the right probe. At the forefront of the components ‘checklist to develop better FI procedural lies the selection of the probe (contrast agent). Unequivocally, the main physics-related drawback for in vivo implementation of this imaging method, as based on light propagation through optical dense media, is lacking enough sub-tissue penetration depth. To address as much as possible that shortcoming, a current top request for any FI probe worth considering is for it to absorb excitation light and to emit its signal within the near-infrared (NIR, 700-2000nm) spectral range. The sub-tissue deep penetration and higher optical contrast achieved by using NIR light of an appropriate wavelength justifies the choice of such radiation. Hence, three biological optical transparency windows (TWs) have been specifically recognized TWI: 700 nm to 950 nm; TW-II: 1000 nm to 1350 nm; TW-III: 1550 nm to 1700 nm). In these TWs, the light absorption and scattering by biological components is minimized making feasible, in the most favorable scenario, up to a few centimetres of penetration depth underneath the skin. The first two aspects make a strong case to markedly increase the optical penetration depth, also mitigating the light scattering that negatively affects optical contrast.

There is a material family of inorganic nanoparticles that has deservedly featured a huge wave of publications on synthesis, characterization and especially biomedical proof-of-concept spanning through imaging, sensing and therapy. That is, the lanthanide-doped luminescent nanoparticles (Ln-LNPs). What exactly these lanthanide-based nanoparticles have to offer as biomedical in vivo probes? Like all inorganic nanoparticles, they are noticeably stable, both chemically and under prolonged photo-stimulation as well. The most characteristic features shown by Ln-LNPs are their spectrally sharp (discreet and narrow) and long-lived (milliseconds down to microseconds) emissions.
The only main limitation of that kind of material? low light absorption.

THE ANSWER: the antenna approach is getting the ""best of two worlds"": the organic dye highly absorbing photons at TW-I, transferring that optical energy to the lanthanide ions (inside the inorganic nanoparticle to which the organic dye is attached) and then the (bright) NIR-II light comes out -featuring all the spectral y temporal assets described in the paragraphs above.

"The WORK PERFORMED during the initial 20 months has led to choose the most suitable lanthanide ion (namely neodymium). Then, a vigorous program of nanoparticles synthesis was launched, -once the proper and adequate set up at chemistry lab of the Partner Organization (Stanford University) was built up. In parallel, a comprehensive careful study on organic dyes absorbing light at TW-I was carried out.

1) On-site synthesis of NaGdF4 (fluoride) neodymium-doped nanoparticles, of a quasi-monodisperse size distribution (centered around 30nm).
2) Selection of the Cyanine7 dye as the most adequate antenna compound, from a multiparametric set of criteria.
3) Obtaining a set of concentration values to optimize the inorganic-organic mixing of the luminescent nanoparticles (acceptor) and TW-I absorbing dye (donor).
4) Evidence was collected of clear intensity enhancement (!) of TW-II emission from several from the preliminary ""joint constructs"" tested.
THE CONTEXT: Cancer is the leading cause of death worldwide and incidence is only expected to increase further due to an aging population and mounting environmental risk factors. In EU-28 area, the estimation is above 1 million people dying in 2018 of cancer and 3 million new cases being diagnosed [ECIS data]. While some cancers have relatively high five-year survival rates if diagnosed when cancer cells remain localized at the primary tumor site and sentinel lymph nodes (SLNs), prognosis is poor for solid tumors that are not discovered before becoming metastatic.Therefore there has been a major emphasis on early screening with the goal of detecting the disease at a stage when it is easier to treat, which is especially important for cancers that are aggressive or asymptomatic in their early forms.
Real-time imaging of tumors during surgery is important to ensure that the tumor be completely excised and thus prevent recurrence while avoiding unnecessary tissue removal that would contribute to morbidity and loss-of-function. Intra-operative imaging can also be helpful for the collection of biopsy specimens for the clinical staging of cancer. For some cancers, including certain forms of breast cancer, the standard of care is SLN biopsy concurrent with surgical resection of the primary tumor. The data obtained from the histological analysis of these samples can serve to inform doctors about the spread of the disease and best course of treatment.

Fluorescence imaging offers high sensitivity, excellent resolution (1 μm) at the tissue surface, and requires relatively inexpensive equipment.

TAKE-HOME MESSAGE: new and cost-effective way of performing biomedical imaging, decisively changing for good the healthcare quality in European countries.
Whole project scheme: the VISION