Development of pharmaceutical compounds intended for diagnostics applications - Exploring the SWIR region

Among the diagnostic techniques for the detection of diseases and, especially, tumours, fluorescence-guided surgery (FGS) has recently emerged as a promising intraoperative technique, that could help surgeons in tumour resection, enabling complete tumour removal and improving surgical outcomes, thus reducing the risk of recurrences. FGS exploits fluorescent probes capable of accumulating within tumour sites to enable the detection and visual localisation of tumour margins, thus facilitating complete resection of the cancer. In recent years, several fluorescent probes have been developed and evaluated for their ability to recognise and accumulate within tumours. Both mechanisms of passive accumulation of the probe in tumour tissue and of targeted delivery to specific tumour types, mediated by binding the fluorescent moiety with tumour-targeting ligands capable of recognising tumour markers overexpressed in tumour tissues, have been explored. The nearly totality of the probes developed, currently under clinical evaluation, are composed of fluorophores emitting in the near-infrared region I (NIR-I, 700 – 1000 nm), mainly belonging to the family of cyanine dyes. The unveiled superiority of optical imaging in the shortwave infrared region (SWIR, 1000 – 1700 nm, also called near-infrared region II, NIR-II), characterised by reduced background autofluorescence, low tissue scattering and deeper penetration of photons into biological tissues, has fostered an in-depth search for suitable SWIR emitters to be used for fluorescence-based in vivo optical procedures. Although many advancements have been made in the development of fluorophores emitting at wavelengths above 1000 nanometres, some drawbacks still limit their application in vivo, such as the substantial hydrophobicity resulting from their highly conjugated structure, which is necessary to allow the emission at such longer wavelengths. Moreover, very few examples of tumour-targeted fluorescent probes composed of tumour-targeting small molecules bound to SWIR-emitting fluorophores are available to date. On these premises, the PhD project summarised in this thesis was devoted to the development of new probes with fluorescence emission in the SWIR region, that could present better compatibility with aqueous environments in view of use for biological imaging, and that could be conjugated to specific ligands directed to tumour markers to widen the field of tumour-targeted fluorescent probes. In Chapter 2, we envisioned that structural modulation of a well-known SWIR emitter, characterised by substantial lipophilicity, through the addition of polar moieties on the fluorophore scaffold, could lead to more water-compatible fluorophores. Therefore, a new series of more polar derivatives has been designed, synthesised and optically characterised. Nanocarrier encapsulation of a promising new derivative has shown favourable results for application in SWIR biological imaging. In Chapter 3 we then sought to develop a new conjugatable fluorescent molecule capable of emitting in the SWIR region, to be used for future derivatisation with tumour-targeted moieties. Finally, in Chapter 4, we developed tumour-targeted fluorescent probes that exhibit SWIR emission and selectively target the tumour microenvironment, thanks to the conjugation of fluorophores with a small molecule inhibitor of Fibroblast Activation Protein (FAP), a tumour marker overexpressed in many solid tumours.