Phd in ANGIO-CARDIO-THORACIC PATHOPHYSIOLOGY AND IMAGING

Thesis title: First steps towards the development of a nanotheranostic model for lung cancer precision medicine: plasmonic photothermal therapy with radiolabeled keratin-coated gold nanoparticles

Abstract Theranostic nanomedicine is emerging as a novel attractive entity, taking advantage on the possibility of integrating imaging and therapy into single multi-functional nanostructures. In latest years, gold nanoparticles (AuNPs) have been extensively investigated, gaining increasing interest as thermo-optical transducers for plasmonic photothermal therapy (PPTT) applications in oncology. By converting absorbed light into heat, AuNPs behaves as nanosources of precisely focused hyperthermia, able to deliver effective thermal damage inducing the death of cancerous cells. Despite the promising results, PPTT with AuNPs still presents several critical issues, mainly related to the selective binding for tumor cells and the need to develop a reliable system of spatial localization. The aim of the research project was to provide the basis of a nanotheranostic model with potential application in lung cancer, by overcoming some of the actual drawbacks impairing the translational advances of AuNPs-mediated PPTT. The functionalization of AuNPs, would enhance their biocompatibility, resulting in prolonged retention time in the bloodstream and higher accumulation in tumor site through a mechanism of passive targeting known as enhanced permeability and retention (EPR) effect. In addition, the development of a reliable nuclear medicine imaging system for AuNPs spatial localization would provide the precise definition of the target volume to be irradiated with laser for PPTT applications. In this project, in first place, a novel generation of functionalized spherical keratin-coated AuNPs (Ker-AuNPs) was synthesized and characterized, with the purpose to enhance the biomimetic properties of conventional AuNPs. Besides the advantages in biocompatibility, due to its chemical structure, the surface-coating by keratin, acting as a bridging protein, has provided AuNPs more functional for following radiolabeling. Technetium-99m (99mTc) has been chosen as radiotracer due to its wide availability from molybdenum-99 (99Mo)/99mTc generators and its ideal physical properties, such as the short half-life (6 h) and the monoergetic gamma-photon emission (140 keV), which are optimal for both high-resolution single photon emission computed tomography (SPECT) imaging and patient safety. A chelator-mediated indirect radiolabeling strategy was pursued in this project by exploiting the well-known metal chelation capabilities of diethylen-triamine pentacetic acid (DTPA). The radiochemical purity (RCP) of the 99mTc-DTPA-Ker-AuNPs obtained following the final and optimized radiolabeling strategy, approached the value of 95% required for radiopharmaceuticals developed for diagnostic purposes. The light-triggered photothermal charachterization of both the radiolabeled nanoplatforms dispersed in saline solution and the radio-instant thin layer chromatography (ITLC)-silica gel (SG) plates, depicted a significant temperature increase (> 58°C), corroborating the presence of perfectly functional 99mTc-DTPA-Ker-AuNPs. The spectroscopic examination of the 99mTc-DTPA-Ker-AuNPs solution supported the occurrence of a novel Ker-AuNPs configuration and confirmed the excellent stability of the obtained compound after 60 days. Further charachterization of 99mTc-DTPA-Ker-AuNPs included the determination of dynamic light scattering (DLS) and z-potential, as well as transmission electron microscopy (TEM) imaging. SPECT imaging revealed uniform radioactivity distribution of 99mTc-DTPA-Ker-AuNPs inside the capillaries and the tubule of a nephron-mimicking chip. A cross-check photothermal experiment performed on the microfluidic device confirmed homogeneous photothermal response. As concerning biocompatibility studies, the administration of 99mTc-DTPA-Ker-AuNPs for 24 h does not produce any crucial effects on inhibiting the proliferation of healthy human embryonic kidney (HEK293T) cells for the concentration range tested (0-100 mM). HEK293T cells incubated with 100 mM 99mTc-DTPA-Ker-AuNPs for 24 h showed normal morphology and preserved ultrastructural arrangement on TEM. PPTT experiments performed with both HEK293T and human cell line (U87-MG) derived from glioblastoma, revealed significant differences in terms of viability reduction, with cancer cells showing more potent thermal cytotoxicity than normal cells. This project provided a novel functionalized and radiolabeled nanotheranostic agent for selective anticancer PPTT. Future active targeting of our 99mTc-DTPA-Ker-AuNPs, would lead the research into the field of precision medicine for lung cancer specific applications.