Establishing the rationale for the design of an enhanced mRNA vaccine based on hybrid nanosystems: use of lipid-coated nanoparticles

According to the World Health Organization, cancer is a large group of chronic, difficult-to-treat diseases with more than ten million new cases each year, and it still remains the second leading cause of death in the world. In addition to mortality, it is also a significant cause of morbidity, due to the problems caused by traditional treatments, such as surgery, radiotherapy, chemotherapy, or a combination of these methods. In recent years, research is strongly focused on finding new therapies that are effective and mostly with a lower incidence of side effects, among the latter, cancer immunotherapy is gaining attraction. Immunotherapies include various approaches among which treatment vaccination with tumor antigens that allows for increased antigen presentation to enhance the ability of the patient's immune system to mount an immune response against neoplastic cells. This project is the result of a collaboration between IQS of Barcelona and the Politecnico di Torino. The IQS group has previously demonstrated the remarkable versatile properties of end-modified poly (β-amino esters) oligopeptide (OM-PBAEs) to complex mRNAs and form discrete nanoparticles that enable specific targeting of antigen-presenting cells (APCs) in vivo. In contrast, at PoliTo, it was demonstrated how lipid nanosystems, composed of lipid-coated inorganic nanoparticles with antitumor properties, increase the cell penetration efficiency, biomimicry, non-immunogenicity, and bio-stability of inorganic nanoparticles. Combining both experiences, we focused on the development and study of non-viral vectors using liposome conjugated pBAE nanoparticles for an enhanced mRNA vaccine based on hybrid nanosystems. For this purpose, gold nanosystems have been first synthesized and characterized, in particular nanospheres and nanorods, which thanks to their inertness and stability allow to better perform the lipid coating following the solvent exchange method protocol. This method is based on the process of preparation of liposomes, called reverse-phase evaporation method, and with appropriate modifications allows to create a lipid membrane to the metal nanoparticles. Subsequently, two variants of the solvent exchange method have been tested on pBAE nanoparticles and characterization methods, such as NTA and CryoTEM, applied to demonstrate their encapsulation. Since the solvent exchange method is a process that uses, albeit in small amounts, ethanol, preliminary studies have been carried out to test the stability of the polyplexes in this solvent. However, considerable difficulties have been encountered with polymeric nanoparticles, which hardly maintained their stability during the encapsulation process and are therefore hardly visible in CryoTEM images. On the contrary, TEM images showed how gold nanosystems well interact with liposomes both internally and externally in small groups. However, the second encapsulation method for polymeric nanoparticles was chosen, which showed the synthesis of large and well-defined liposomes in which they are expected to contain the nanoparticles. Subsequent steps of purification by sonication and filtration have been done to allow obtaining small size and monodisperse nanosystems. Finally, preliminary in vitro transfection studies were performed on tumor cell line, chosen as a model, that showed an increase in transfection efficiency for liposome-conjugated nanosystems.