EARLY DEVELOPMENT OF AN INNOVATIVE NANOPARTICLE-BASED MULTIMODAL TOOL FOR TARGETED DRUG DELIVERY: A STEP-BY-STEP APPROACH

Prostate cancer is the most common tumour in men in developed countries and, often, it responds poorly to conventional treatments such as surgery, radiation or androgen deprivation. Monoclonal antibody (MoAb) therapy, for this kind of pathology, has grown tremendously in the past decades exploiting antibodies in their naked form or conjugated to cytotoxic payloads to form antibody drug conjugates (ADCs). In particular, prostate cancer is featured by the high expression of the prostate-specific membrane antigen (PSMA) which is an internal cell membrane 100 kDa glycoprotein already validated as a pathology marker due to its restriction in normal tissues. Several studies have been carried out conjugating biomolecules against PSMA to cytotoxic drugs such as monomethyl auristatin E (MMAE). Nano-based formulations are representatives of another technology with high potential in targeted drug delivery to enhance the bioavailability of drugs and, consequently, enhance the therapeutic window. Our research aimed to evaluate if fluorescent PEGylated silica nanoparticles, already optimized for in vitro immunoassays, might be used in selective cancer cell targeting and killing. The nanoparticles employed in this study were featured by a core-shell structure allowing the contemporary conjugation of the targeting molecule (anti PSMA antibody) and the cytotoxic payload (MMAE), thanks to the intrinsic fluorescence of the nanoparticles the fate of the construct might be monitored across time. The experimental approach chosen is a step-by-step one in which all the components of the final multimodal tool were designed, produced, proved for lot-by-lot repeatability and reproducibility and, successively, singularly tested on cellular models in three independent sessions. Once outlined their stand-alone performance a small new brick was added in the building of the final compound. We observed that stand-alone nanoparticles were actively, rapidly and spontaneously internalized by cell lines without affecting their health state. We analyzed their intracellular localization, finding a specific tropism for mitochondria. We analyzed their intracellular localization, finding a specific tropism for mitochondria. Secondly, we conjugated nanoparticles to doxorubicin observing the conserved capability of the system to be internalized but, contemporary the partial loss of a satisfactory cytotoxic effect, probably due to the lack of a complete and efficient release of the active principle. Of note, the system seems to offer the advantage of reducing the release of CD44+ Extracellular vesicles. Subsequently, we conjugated the nanoparticles to anti PSMA antibody, and as the steric hindrance due to the presence of nanoparticles was significantly higher than the one due to the sole drug, we parallelly evaluated the same antibody linked to fluorescein, a small fluorescent molecule with a size comparable to those of the small drugs. After the assays on cells, we stated that no significant differences in fluorescence signal were attributable to the difference in size. In addition, thanks to a competitive analysis we confirmed the receptor-mediated nature of the endocytosis observed. Finally, we obtained the multimodal tool by conjugating the antibody to MMAE and then the complex to nanoparticles. We compared its cytotoxic effect near to the antibody-MMAE and free MMAE. We found a lower cytotoxicity effect of the nanoparticle-based construct concerning free drug, likely because of the preservation of the previously observed receptor-mediated endocytosis. The results collected in this study confirmed nanomedicine as a powerful alternative to organic drug delivery systems. However, before silica can be routinely introduced in clinical settings[, some aspects, such as drug loading efficacy, spatial and temporal drug release, scalable manufacturing and long-term stability, need to be deepened.