Phd in MATHEMATICAL MODELS FOR ENGINEERING, ELECTROMAGNETICS AND
NANOSCIENCES

Thesis title: Novel liposome formulations for brain delivery

Neurological diseases are a major medical emergency, and despite efforts to implement new therapies, treatment is severely hampered by the inability of neuropharmaceuticals to cross the blood-brain barrier (BBB). Therefore, since drug delivery, rather than drug efficacy, is the crucial issue in the treatment of central nervous system diseases, the development of suitable nanocarriers capable of crossing the BBB and delivering the drug to target districts is a primary goal of harmaceutical research. Engineered nanoparticles in nanomedicine are widely studied for their potential use as drug delivery systems due to their ability to distribute themselves throughout the body and the possibility of engineering them for multiple purposes. In the context of nanocarriers, liposomes represent ideal drug delivery systems because their characteristics can be easily and adequately regulated, they usually exhibit low toxicity as well as biocompatibility and biodegradability. In this scenario, the aim of the project was to develop new liposome formulations for the treatment of diseases of the CNS. The investigation focused on the development of liposomes functionalized to be able to cross the BBB, load high quantities of drug and deliver it to the CNS. The formulations investigated were composed of a natural phospholipid (DMPC, DPPC, DOPC) and cholesterol (CHOL) mixed with cationic amphiphiles synthesized within the scope of this work to promote the ability of the liposome to cross the BBB. Two types of cationic amphiphilic structures were selected: (1) the diastereomeric gemini amphiphiles SS and MESO (Chart 1), those structure should confer a certain stability to the liposome bilayer and at the same time promote the crossing of the BBB via adsorption-mediated transcytosis; (2) the glycosylated amphiphiles MAN1, GLT1, GL4 (Chart 1), characterised by a glycosyl residue, which should promote crossing via carrier-mediated transport (in this specific case by GLUT1). Formulations including the SS amphiphile were also prepared in the presence of a pegylated phospholipid (mPEG-DSPE), in order to compare non-pegylated and pegylated formulations in terms of stability over time and ability to cross the BBB. In addition, doubly functionalized liposome formulations consisting of a cationic gemini amphiphile and a glycosylated lipid (MAN1, GLT1, MAN-PEG3-PE, Chart 1) were also studied. The novel formulations, designed as potentially suitable for crossing the BBB, were all analysed with the aim of investigating how the different stereochemistry of the gemini or the presence of glycosyl residues may influence the physicochemical and biological characteristics of the final liposomes. In particular their potential was evaluated from two different perspectives: 1. to verify the ability of the liposomes to cross the BBB and the effect of functionalization: for this purpose, an internal probe was inserted into the formulations as a tracer to follow the biological experiments on human-derived in vitro BBB models developed by IRBM SCIENCE PARK with whom this work was in collaboration. Different molecules were explored as tracers of liposomes, such as kynurenic acid (KYNA), which is trapped in the aqueous core, or resveratrol (RSV), which is inserted in the bilayer; 2. to test the ability of the formulations to efficiently transport a drug of neurotherapeutic interest and verify the in vitro efficacy of the drug included in the liposome: in this case, DMPC/gemini liposomes were studied for their ability to complex and deliver to motor neurons an antisense oligonucleotide (ASO), Nusinersen, recently approved for the treatment of a neurodegenerative disease, spinal muscular atrophy (SMA). Due to Nusinersen's inability to cross the BBB, the drug is currently administered by intrathecal injection. Therefore, the development of a non-invasive drug delivery system is a key goal of the therapeutic approach to SMA. Regarding the first objective of this work, liposome formulations were fully characterised in terms of size, polydispersity, ζ-potential, transition temperature (Tm) and stability over time by dynamic laser scattering (DLS), electrophoretic mobility measurements and fluorescence spectroscopy. The entrapment efficiency of the probes and release over time were assessed by HPLC and UPLC measurements. The overall composition of the formulations was modulated in order to obtain nanosystems of adequate size (100-200 nm) and polydispersion index (PDI), stable with respect to aggregation phenomena and capable of loading and retaining an adequate amount of tracers. This extensive work led to the selection of different liposome formulations whose ability to cross the BBB was studied in biological experiments on the 2D transwell model, consisting of differentiated cerebral microvascular endothelial cells derived from human induced pluripotent stem cells (hiPSC-derived BMECs or iBMECs) co-cultured with human astrocytes. The results of the biological investigations indicate that the composition and physicochemical characteristics of the formulations as a whole, such as charge, size and fluidity, affect the biological response. The presence of gemini promotes the crossing of the BBB and the permeability of the particles depends on the stereochemistry of the gemini. As a final part of this investigation, during the period spent abroad at the Department Nanomedicine and Drug Targeting of the University of Groningen in the group of Prof. A. Salvati, the internalization mechanism of liposomes coated with different glycosylated ligands (GLT1, MAN1, GL4, MAN-PEG3-PE, Chart 1) was studied by using pharmacological inhibitors of GLUT1 transporter (BAY 876 and Phloretin). Uptake studies were carried out, evaluating the internalization of different nanocarriers in Hela cells by cytofluorimetry measurements. Concerning the second objective of this project, with the aim of developing a nanosystem able to deliver Nusinersen to the motor neuron by crossing the BBB, the ability of DMPC/gemini liposome formulations to complex ASO, giving rise to stable formulations, was investigated as a function of DMPC/gemini ratio and as a function of liposome/ASO ratio. The most stable lipoplex formulations were tested in biological studies to evaluate: 1) the in vitro therapeutic efficacy of the nanosystems on primary fibroblasts of SMA patients 2) their internalization in the human-derived BBB model The results highlight that all selected lipoplex formulations were internalised into the BBB model and further studies are in progress to assess the actual permeability through the BBB. Moreover, the lipoplex formulation composed of DMPC/SS complexed with ASO showed an in vitro therapeutic efficacy comparable to that of the free drug. In contrast, the formulation with DMPC/MESO, i.e. with the other diastereomer, was not able to promote exon correction. Overall, the results of this work clearly show how the physicochemical characteristics of the final nanosystem influence its biological fate. Subtle changes in the composition may result in different biological behaviour and, in particular, the stereochemistry of the components can dictate the biological outcome.