Design, optimization, characterization and performance of nanocarriers for brain delivery of natural compounds

The aim of my PhD project was the design, optimization, characterisation and performance of nanocarriers for brain delivery loaded with natural compounds. Blood brain barrier (BBB) represents an important physio-chemical obstacle for the delivery of therapeutics into the brain and the limitations of conventional therapies increase the interest regard the use of nanotechnologies for neurodegenerative treatments. Several natural products have been studied for the prevention and the treatment of brain diseases, frequently, with very interesting therapeutic effects but their poor solubility, bioavailability and BBB permeability have limited their use. To overcome these problematical aspects, different NPs that target and reach brain tissues were formulated and examined during this PhD thesis. My investigation started from the analysis of BBB in order to establish formulative strategies. Two different endogenous mechanisms of transport across the BBB, and deliver therapeutics into the brain, were selected: receptor mediated endocytosis by Apo-lipoprotein E (Apo-E) and adsorptive transcytosis by plasmatic proteins. One synthetic unit, ethyl-cyanoacrylate, and a protein, human serum albumin, were selected as materials for the design of brain delivery systems. In case of albumin nanoparticle, two distinctive method of preparations were investigated in order to reduce the use of toxic agents in the formulation, according with green chemistry criteria. Nanocarriers were developed and fully characterized in term of size, superficial charge, encapsulation efficiency, loading capacity and morphology, using Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). The brain uptake of fluorescent NPs, their biodistribution and cellular-functional effects were evaluated in vivo in healthy rats, after intracerebral injection, while the ability of developed nanocarriers to cross BBB, after intraperitoneal and intravenous administration, were assessed. Behavioural studies were performed for the investigation of safety profile. After the results obtained from the in vivo examinations, Salvianolic acid B (SalB) isolated from Salvia miltiorrhiza Bge. and Andrographolide (AG) from Andrographis paniculata’s leaves were selected, for their pharmacological profile, to be loaded in developed NPs for the treatment of neurodegenerative disease. XX The permeability across the BBB of AG in the free form and loaded in nanocarriers was evaluated using a well established in vitro BBB model based on monolayers of human immortalized endothelial cells hCMEC / D3, that expresses the receptors for Apo-E (LRP-1expression).