Development of targeted stimuli-responsive nanoparticles for combined colorectal cancer treatment

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the world, right after lung cancer. Current conventional treatments do not guarantee complete effectiveness and present significant side effects. Nanomedicine has progressively gained ground in cancer therapy by enabling targeted and more effective treatment with reduced adverse effects due to its ability to interact directly at molecular level. Moreover, recent studies have revealed that tumor treatment with specific classes of nanoparticles (NPs), in combination with external acoustic waves stimulation, results in enhanced therapeutic efficacy. Based on these premises, this thesis project focuses on the development of biomimetic targeted NPs, capable of responding to an external stimulation in the form of shock waves (SW), for targeted treatment of CRC. In detail, iron-doped zinc oxide NPs (Fe:ZnO NPs) were synthesized using a wet chemistry method and subsequently coated with a self-assembled biomimetic lipid shell through the solvent exchange method, to increase biocompatibility, conjugation efficiency and prevent immune recognition. To achieve an active targeting strategy, the lipid shell was functionalized with the YSA peptide (whole amino acid sequence YSAYPDSVPMMS), highly selective for the EphA2 receptor, which is markedly overexpressed in CRC cells. Particular attention was given to the optimization of the peptide conjugation process. Two different conjugation protocols were tested, and then the ratio and amounts of peptide and lipid were investigated, both evaluated by fluorescence microscopy to optimize conjugation efficiency. With the aim of studying the effect of the targeting agent on NP uptake and cytotoxicity induced by combined application of SW and NPs, a comparative study was performed by administering NPs without targeting peptide (L-Fe:ZnO NPs) and with the targeting peptide (YSA-L-Fe:ZnO NPs) on 3D organoid models of patient-derived metastatic CRC cells, provided by the XENTURION biobank (IRCCS Candiolo, Torino), and on the healthy counterpart (CCD-18Co spheroids). The study showed significant internalization of both NP types in CRC organoids, demonstrating a certain selectivity towards CRC cells, and, in addition, pronounced mortality was observed in treated CRC organoids after SW application. In parallel, an artificial intelligence (AI) algorithm, developed by M.D. Eng. Alessandro Casella, was employed due to its ability to distinguish regions containing CRC cells (HT-29) from regions containing healthy cells (CCD-18Co) in 2D cocultures subjected to two different treatments: one case with L-Fe:ZnO NPs, and the other with YSA-L-Fe:ZnO NPs. This methodology enabled monitoring the evolution of cocultures over time to compare the effects of treatments on HT-29 regions. In general, this project highlights that the combined approach involving biomimetic stimulus-responsive NPs and external stimulation by SW represents a promising strategy for the selective treatment of CRC. Thus, this work provides the basis for further studies to validate and possibly extend this combined strategy to other tumor types, contributing to the development of more specialized, safe and effective therapies. Lastly, the use of AI for precise monitoring of the treatment effect over time offers a versatile tool potentially applicable to other cancer treatments.