Superparamagnetic nanoparticles loaded in red blood cells as novel contrast agents for in vivo diagnostic techniques in the biomedical field.

Nanomedicine has revolutionized biomedical research by integrating diagnostic and therapeutic functionalities into novel platforms. This thesis explores the engineering of red blood cells (RBCs) as biocompatible carriers for superparamagnetic iron oxide nanoparticles (SPIONs), offering a groundbreaking approach for in vivo imaging and drug delivery applications. RBCs provide a unique advantage due to their long circulatory lifespan, immune evasion properties, and capability to encapsulate various nanomaterials, thereby enhancing the stability and efficiency of contrast agents used in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). The research focuses on optimizing the synthesis of SPIONs through a multi-step process, incorporating advanced surface coating techniques with citrate and dextran to improve monodispersity, stability, and encapsulation efficiency. The encapsulation of SPIONs into RBCs was achieved by transiently opening membrane pores via reversible hypotonic hemolysis, ensuring nanoparticle internalization while maintaining cell viability. Among the synthesized nanoparticles, formulations such as MNPs 29DX-filt 0.1 and MNPs 40 DX-filt 0.1 demonstrated superior performance in both human and murine RBCs. Comprehensive characterization techniques, including Transmission Electron Microscopy (TEM), Nuclear Magnetic Resonance (NMR), Magnetic Particle Spectroscopy (MPS), and preclinical MRI at 1 Tesla, were employed to assess the physicochemical properties and imaging potential of these constructs. Notably, MPS, an emerging technology linked to MPI, revealed enhanced tracer quantification capabilities, reinforcing the suitability of SPION-loaded RBCs for high-sensitivity imaging applications. This work also investigates the potential of RBC-encapsulated SPIONs as an alternative to conventional blood oxygenation level-dependent (BOLD) contrast agents for functional MRI (fMRI). The use of Ferucarbotran®-loaded RBCs (FLH-RBCs) for cerebral blood volume (CBV)-weighted fMRI demonstrates improved spatial specificity and prolonged circulation compared to free SPIONs, offering a transformative approach for functional brain mapping. Preclinical studies in rodent models validated the efficacy of this novel imaging strategy, showing superior cortical layer resolution over traditional BOLD-based techniques. Furthermore, a key aspect of this research is the integration of automation trials in the RBC-loading process, conducted in collaboration with EryDel S.p.A. The development of an automated system for Ferucarbotran® encapsulation could ensure reproducibility, scalability, and clinical feasibility, overcoming challenges associated with manual loading methods. Beyond imaging, the thesis explores the compatibility of nucleobase-containing platinum(II) complexes with RBCs for potential anticancer and antiviral drug delivery. Encapsulation studies demonstrated that RBCs effectively protect these therapeutic agents from rapid degradation, ensuring controlled release and improved biodistribution. The feasibility of loading various metal-based nucleoside derivatives into RBCs opens new avenues for advanced targeted therapies. Overall, this work highlights the remarkable versatility of RBC-encapsulated nanomaterials for biomedical applications, offering innovative solutions for molecular imaging, targeted drug delivery, and functional neuroimaging. By leveraging the unique biophysical properties of RBCs and state-of-the-art nanotechnology, this research paves the way for the development of next-generation diagnostic and therapeutic strategies.