Study of Protein Corona effect on different biomimetic Nanoparticles' formulations for cancer targeting and therapy

Nanoparticles (NPs) have been gaining growing interest in the biomedical scene thanks to unique characteristics such as high surface-to-volume ratio and tuneable surface chemistry, that allows them to be a first-line-strategy for cancer treatment, overcoming limitations related to the patient-to-patient variability and off-target side effects. NPs can be categorized based on the constitutive material, identifying polymeric, inorganic and lipid-based NPs, each one with his own pros and cons regarding cargo, delivery and related immune-response. Additionally, hybrid nano-systems are increasingly being studied to create biomimetic NPs with enhanced characteristics in terms of cell targeting and chemical stability in biological environment. When in contact with biological fluids, such as blood, plasma or serum, protein absorption takes place on the surface of the NP, forming what has been defined as protein corona (PC). Initially, the most abundant proteins with lower affinity for NP surface compose a first layer called soft corona. Over time, this layer evolves into a more stable structure with tightly bound proteins, forming the hard corona. PC formation on NPs can significantly alter their stability and biodistribution, conferring them a new biological identify. Differences in PC composition have been observed depending on NPs composition, curvature and environmental conditions, underlining the need for detailed characterization. To limit the effects caused by PC formation, NPs have been engineered with biomimetic lipid coatings containing cholesterol and phospholipids, mimicking the main components of the cell membrane. The aim of this Thesis is to explore PC formation on pristine iron-doped zinc oxide NPs (Fe6:ZnO), silica (SiO2) NPs and their counterparts coated with different biomimetic lipid formulations. Fe6:ZnO NPs were synthetized using a wet chemistry technique with oleic acid as capping agent, further functionalized with aminopropyl group to impart a positive surface charge. In turn, SiO2 NPs were synthesized following Stöber method and acquired a negative surface charge. The synthesized NPs were characterized in terms of size, morphology and surface properties and then coated with lipids using solvent-exchange technique. In particular, the lipid formulation used to coat ZnO NPs was inspired by COVID-19 vaccines, combining phospholipids and cholesterol along with a PEGylated lipid conjugated with a targeting peptide. This lipid coating showed an overall negative charge to induce an electrostatic interaction with the ZnO surface. The lipid coatings of SiO2 NPs were instead based on positively charged lipids and the composition adopted has a fusogenic attitude towards the cells, ultimately allowing investigation of two different mechanisms of cell internalization. NP interaction with proteins, was investigated upon incubation in cell culture media, supplemented with fetal bovine serum, and in human plasma, by evaluating the subsequent change in the surface charge, giving insights into PC formation. Protein absorption was assessed through Bradford assay and gel electrophoresis. Finally, this work investigated the influence of the PC on the biological behaviour of two types of inorganic NPs, with a particular focus on the impact of different biomimetic lipid coatings. The findings contribute to a better understanding of the potential of biomimetic surface modifications, offering valuable insights for the future development of effective nanosystems for cancer therapy.