Dottorato in FISIOPATOLOGIA ED IMAGING CARDIO-TORACO-VASCOLARE

Titolo della tesi: Gold nanoparticles labelled with an alpha emitter: a theragnostic double therapy agent for Non-Hodgkin Lymphoma and Paragranuloma

Background: The use of gold nanoparticles (AuNPs) in theranostics represents a thrilling and novel perspective for oncology. AuNPs can induce cell death through the so-called “plasmonic photothermal therapy” (or PTT). It consists of the conversion of the incident laser beam light into heat, inducing cell death. In this study, double-layer keratin gold bipyramid nanoparticles (DL-Ker-AuBPys), single-layer keratin gold bipyramid nanoparticles (SL-Ker-AuBPys), and gold nanorods (AuNRs) were evaluated in terms of toxicity, biocompatibility, and were labelled with Radium-223 (223RaCl2, radium). Gelatine-Na2HPO4 and DTPA were evaluated as possible chelating agents able to improve radiolabeling with radium. The functionalization with the mouse monoclonal antibody 1011:sc-57709 was then explored to obtain target-specific nanoparticles. This work aimed to develop a potential theragnostic agent useful to achieve therapeutic radicality in Non-Hodgkin lymphoma (NHL) and nodular lymphocyte-dominant Hodgkin lymphoma (NLPHL) in association with radio-chemotherapy. This could be realized by combining the therapeutic features of 223RaCl2 and AuNPs with the gamma emission of 223RaCl2. Methods: Stable colloidal AuBPs were prepared through a seed-mediated growth approach and were functionalized with keratin. U87-MG cells were used to evaluate the biocompatibility of AuBPs through a trypan blue exclusion assay. After the incubation with AuBPs, the evaluation of U87-MG cell viability was determined through laser illumination: U87-MG cells (5 x 105) were incubated for 24 and 48 h with 100 μM DL- Ker-AuBPs and were illuminated for 6 minutes. In the second part of the work, DL-Ker-AuBPs were incubated with 0.25 ml of 223RaCl2 (activity of 0.9 MBq in 1.6 ml) for 5, 15, and 30 minutes at room temperature. Then, a total quantity of 0.5 ml DL-Ker-AuBPys was placed vertically in a shielded water bath at 45°C, 0.25 ml of 223RaCl2 was added, and samples were taken at 0, 5, 15, and 30 minutes and after 20 minutes of cooling at room temperature. Instant thin-layer chromatography (ITLC) was conducted for strips with DL-Ker-AuBPys, 223RaCl2-DL-Ker-AuBPys, and 223RaCl2 alone. A Perkin Elmer Lambda 365 spectrophotometer was used to acquire reflectance and absorption spectra of strips and of DL-Ker-AuBPys and 223RaCl2-DL-Ker-AuBPys solutions to study if an effective radiolabelling had occurred. The strips were examined by a thermal scan with an 808 nm light beam to establish the precise localization of the nanoparticles. The pH value of 223RaCl2-DL-Ker-AuBPys solution was measured after 30 minutes of incubation at room temperature and post-cooling after 30 minutes of incubation at 45°C. To better the labelling with 223RaCl2, gelatine-Na2HPO4 and diethylenetriaminepentaacetate (DTPA) were evaluated as chelating agents: absorbance spectra were acquired for 223RaCl2-DTPA-NRs and 223RaCl2-gelatine-Na2HPO4-AuNRs solution to demonstrate the functionalization of gold nanocarrier with the chelating agent. ITLC was performed only for 223RaCl2-DTPA-NRs. In the last year of the study, AuNRs were functionalized with PAH (polycyclic aromatic hydrocarbons) and bioconjugated with mouse monoclonal antibodies 1011:sc-57709. Absorbance spectra of AuNRs and AuNRs/PAH/Ab solution were performed to determine the effective functionalization of AuNRs. Results: DL-Ker-AuBPys demonstrated a high degree of biocompatibility. Conversely, SL-Ker-AuBPys were toxic for U87-MG cells starting from a concentration of 50 μM at 24h and 10 μM at 48 and 72h. DL-Ker-AuBPys greatly induce cell death in U87-MG cells when they are not washed out before the illumination. The labeling yield of 5 minutes of incubation of 223RaCl2 with DL-Ker-AuBPys at room temperature was 7.8%. No significant changes in the labeling yield or in the pH value (equal to 6) were found when incubation time and temperature were modified. However, the reflectance spectra of the strips dipped in saline, respectively with 223RaCl2-DL-Ker-AuBPs and DL-Ker-AuBPs, suggested effective radiolabeling. It was also confirmed by the absorption spectrum of 223RaCl2-DL-Ker-AuBPs solution. Thermal scanning of the strips with 223RaCl2-DL-Ker-AuBPs and DL-Ker-AuBPs showed localized heating around the center where the drop was deposited, confirming the presence of NPs. DL-Ker-AuBPs demonstrated high viability when incubated for 24 and 48 hours with U87-MG cells. Gelatine-Na2HPO4 was shown to be a possible candidate as a chelator agent, but only decayed 223RaCl2 was used for this functionalization experiment. DTPA betters the labelling of AuNRs, demonstrating to be an excellent chelating agent for the radiolabeling of AuNRs. Finally, the use of PAH permitted the functionalization of AuNRs with antibodies, creating a novel theragnostic tool feasible for the treatment of LNH and NLPHL micrometastases. Conclusions: DL-Ker-AuBPs are biocompatible nanocarriers with great photothermal capacity to induce cellular death. They were successfully labelled with 223RaCl2, but with low label efficiency, which is not improved by modifying incubation time and temperature. AuNRs have been demonstrated to be a better candidate for theragnostics thanks to their easier labelling and functionalization with antibodies. However, further studies are required to better evaluate the feasibility of use in clinical trials.