Resumen de Estudi de nous liposomes per a l'administració d'enzims a través de membranes biològiques
Liposomes are lipid-based nanovesicles widely explored as nanocarriers for the transport of biomolecules or drugs of interest to the place of action and for the development of new nanomedicines. This Thesis is devoted to the study of liposomal systems functionalized with targeting-ligands, with the final goal to be used as nanocarriers of therapeutically active enzymes. The new liposomal formulations have been specifically investigated and developed for the effective transportation of α-galactosidase A enzyme through cellular and blood brain membranes, and for the achievement of a new liposomal intravenous pharmaceutical product for the treatment of Fabry disease.
Fabry disease is a rare disease which belongs to the group of lysosomal storage disorders, currently without a definitive cure. It characterizes by the deficiency in α-galactosidase (GLA) enzyme activity which results in the cellular accumulation of neutral glycosphingolipids (mainly Gb3) with multisystemic organ affectation, such as kidneys, heart, and nervous system. The current treatment is the enzyme replacement therapy, in which free GLA recombinant protein is administered intravenously to patients. This treatment shows several drawbacks including poor biodistribution, low stability, limited efficacy, high immunogenicity, and low capacity to cross biological barriers, such as cell membranes and the blood-brain barrier. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating enzymes.
Nanoliposomes functionalized with RGD-peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, this initial GLA-nanoconjugate was still far from the preclinical testing. Several issues must be addressed for transforming this initial GLA-nanoformulation (or nanoGLA) into a pharmacological product. To achieve this transformation, a deep understanding and control of the nanoliposomal vehicle at the molecular and supramolecular level is unavoidable. In this Thesis, the relation between the physicochemical properties and biological behavior of targeted liposomes have been addressed, to get knowledge and gain control of liposomal systems for enzyme delivery, especially for the delivery of GLA enzyme for Fabry disease treatment.
Small and uniform nanoGLA liposomes, functionalized with targeting-peptides, were successfully prepared by DELOS-susp, showing high GLA entrapment efficiency, enhanced enzymatic activity, and superior efficacy. NanoGLA formulation for the treatment of Fabry disease was successfully optimized with the required amount and quality to advance towards the preclinical evaluation in vivo. NanoGLA demonstrated superior efficacy compared to current treatment (Replagal®) and non-nanoformulated GLA in a Fabry KO mouse model in terms of higher reduction of Gb3 levels in all tested tissues, including brain. Further, the regulatory preclinical stage of development for this novel nanoGLA formulation was achieved for first time with GLP toxicity studies in rodent. Finally, alternative new ligand-targeted functionalized nanoliposomes prepared by the CO2-based DELOS-susp technology were explored for blood-brain barrier crossing applications.
In summary, the results achieved in this Thesis support the strong potential of targeted liposomal systems for nanomedicine and drug delivery application. The successful development and optimization of the nanoGLA product for improving the current enzymatic replacement therapy in Fabry disease especially contributes as an example of translational and interdisciplinary research.
