Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: PET and MR Thermometry imaging–guided photothermal therapy assisted by radiolabeled keratin-coated gold nanoparticles for precision anticancer thermoablation

Nanomedicine is an emerging key technology of the 21st century. Gold nanoparticles (AuNPs) have been actively investigated as photothermal converters, drug delivery systems, radiosensitizers, and molecular imaging probes in a wide range of biomedical applications in both cancer diagnostics and therapeutics. The flourishing development of AuNPs applications has led to the opportunity of a new targeted cancer treatment that could be capable of selectively fighting cancerous cells, keeping low harmful side effects suffered by surrounding healthy cells, namely the plasmonic photothermal therapy (PPTT). The PPTT approach should have the following key features: targeted AuNPs delivery to the tumor for effective thermal damage response under NIR laser exposure, aiming to keep the surrounding tissues healthy and accurate thermal dose calculation with non-invasive real-time monitoring of thermometric response profile in the target volume. What constitutes a technological frontier is the application of a novel AuNPs generation properly labeled with a radiotracer which may serve as a nuclear imaging-guided high-precision cancer probe. The topic of this thesis is part of a wider funded project (NANO-TAFT), focused on the development of a novel radiolabeled AuNPs-mediated selective PPPT cancer treatment, supported by the development of a prototypal multimodality PET/MR imaging integration console, using MR Thermometry (MRT) to provide a volumetric "heat map". The aims of my research work were to obtain accurate modeling of PET performance variability in detecting radiolabeled AuNPs and their concentration through a PET imaging phantom study carried out on three different PET systems, and the AuNPs radiolabeling, its further characterization, and nuclear imaging study. The evaluation of the variability of the efficiencies and of the AuNP concentration for each PET machine was done through the study of a phantom model and the standardized uptake value (SUV) calculation, which resulted in PET imaging poor reproducibility in terms of quantitative analysis and determination of concentrations. SPECT is known to be a more versatile and reliable imaging technique, thus it was chosen to use technetium-99m (99mTc) as labeling radionuclide along with SPECT technique, instead of fluorine-18 (18F) with PET imaging. A critical issue must be identified in the achievement of an effective AuNPs radiolabeling, able to demonstrate optimal radiochemical yield and stability performances. A novel bridging molecule, namely keratin, was used to solve the problem of binding AuNPs and radioisotopes. To our knowledge, keratin protein remains still unexplored in the field of PPPT-based applications. A reliable and reproducible procedure for the 99mTc radiolabeling of this novel generation of AuNPs (Ker-AuNPs) was therefore achieved. Optical, spectroscopic, and morphological analysis of the Ker-AuNPs proved their stable bioconjugation. The optimal radiolabeling strategy to obtain 99mTc-KerAuNPs and its relative conditions have been found and validated, leading to an excellent performance in terms of radiolabeling efficiency, that reach over 95% of radiochemical purity (RCP). UV-Vis absorbance test supported the occurrence of a novel Ker-AuNPs configuration. The laser-triggered photothermal test demonstrated a good thermometric response through a significant temperature increase in the site of 99mTc-Ker-AuNPs deposition, while corroborating the successful outcomes of the Ker-AuNPs radiolabeling procedure. SPECT imaging study showed a good performance. The analysis of PET imaging data on phantoms showed that PET is not sufficiently selective in terms of achievable spatial resolution and in the determination of the concentrations, thus its application in the context of this project has proven to be unfeasible. SPECT technique allows a less variable determination of radioisotope concentrations than PET and also a less complex calibration of SPECT equipment. Unfortunately, if reasonably small tumors are considered, both techniques are not able to ensure a proper high spatial resolution, which is a crucial prerequisite for a selective and safe treatment. Despite open issues related to the imaging applications remain unsolved, nuclear imaging techniques will be useful rather to determine the targeted volumes of interest, which subsequently will be more accurately confirmed by MR imaging, which has the absolute advantage of a better spatial resolution. A novel functional and stable AuNPs keratin coating has been provided, leading to a promising novel tool in the field of biocompatible photothermal nanoagents for selective anticancer PPPT treatments.