Well-defined polyglutamates as carriers for the treatment of neurodegenerative diseases
- Autores: Aroa Duro Castaño
- Directores de la Tesis: María J. Vicent Docon (dir. tes.)
- Lectura: En la Universitat de València ( España ) en 2015
- Idioma: inglés
- Tribunal Calificador de la Tesis: Cameron Alexander (presid.), Blanca Vázquez Lasa (secret.), Giuseppe Battaglia (voc.)
- Materias:
- Enlaces
- Resumen
Alzheimer’s disease (AD) is a neurodegenerative multiple process of the central nervous system, which currently represents the most common cost of Dementia. The already high incidence of AD is predicted to dramatically increase over the years. In fact, the experts claim that it will become a global epidemy by 2050. Consequently, direct and indirect costs related to AD are doomed to dramatically increase. For instance, only in America, AD related burden will overcome the trillion of dollars by 2050. Moreover, available medication (Exelon®, Namenda®, Aricept®, and Razadyne®) produce moderate symptomatic benefits, but do not stop disease progression. Hence, AD, among other neurodegenerative disorders, can be considered an unmet medical need. Neuroprotective drugs, such as, curcuminoids are been taking in high consideration in order to approach these fatal disorders from a protective and preventive point of view. In this context, nanomedicine and, in particular, Polymer Therapeutics (PT) emerge as a powerful alternative to overcome the limitations of low MW drugs including their poor pharmacokinetic and pharmacodynamic profiles and low solubility in aqueous solvents, required for i.v. administration. Nonetheless, in the PT field, there is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Apart from biodegradability, the development of novel well-defined architectures with higher MW (in order to increase passive targeting provided by the EPR effect), predictable structure and conformation (defined three-dimensional architecture in solution), higher homogeneity, greater drug loading capacity and increased multivalency is considered crucial. To this respect, polypeptides are envisaged to achieve a major impact on a number of different relevant areas including nanomedicine. Thus, new PT based on amino acids are excellent candidates for drug delivery, as they do not suffer from the previously mentioned limitations. Concretely, polyglutamates constitute a versatile platform, which has been effectively used as building blocks in polymer drug conjugates and polymeric micelles for various medical applications ranging from cancer to regenerative medicine. Moreover, it is expected its FDA approval after approval of PGA-paclitaxel conjugate, OpaxioTM for the treatment of various cancers alone or in combination (OpaxioTM has been recently designated as orphan drug in combination with radiotherapy and temozolomide for the treatment of glioblastoma multiforme). Nevertheless, control on polymer chain length, polydispersities and stereochemistry has been the major challenge in the development of synthetic polypeptides over the past years. Besides, the use of branched polymers is emerging in order to accomplish the previously described requisites. They exhibit special properties when compared to their linear counterparts. As a result of their different architectures, solution conformation, size and shape as well as greater multivalency, different therapeutic outputs could be gained. Due to their compact and globular shapes they are postulated to perform better regarding to overcome biological barriers, a pre-requisite in neurodegenerative disorders treatment as well as diagnostics due to the presence of the blood-brain barrier (BBB), one of the most challenging to surpass. Therefore, the main aim of this thesis was the design of new versatile polyglutamate-based nanotherapeutics to be used in the treatment and/or diagnosis of devastating neurodegenerative pathologies such as AD. In order to accomplish our final goal, firstly, we report the development of synthetic pathways to a plethora of functional polyglutamates with well-defined structure, adjustable MW and low polydispersities (? <1.2) applying the ring opening polymerization (ROP) of N-Carboxyanhydrides (NCA) with novel initiators. Furthermore, this methodology has been extended to reach a number of architectures based on PGA, including stars, grafts, and hybrid di-block copolymers. In addition, a versatile post-polymerization modification method to introduce a variety of functionalities such as alkyne, azides, reactive disulfides, maleimide groups or protected amines has been developed, yielding a set of orthogonal reactive attachment sites suitable for further bioconjugations. The physico-chemical properties of the obtained polyglutamates have been exhaustively investigated, in terms of size and solution conformation by the use of a battery of complex techniques including DLS, DOSY-NMR, CD, TEM and SANS. Furthermore, we have developed a novel PGA-based family of systems that, according to their physico-chemical characterization, underwent a self-assembly process where it did exist a structure/conformation-concentration dependency encountering at low concentrations “unimers” of 5-10 nm size, whereas bigger structures of around 100-180 nm were formed at high concentrations. After covalent entrapment of these bigger structures by means of click chemistry, the concentration dependence conformation was clearly eliminated. We have taken profit from that special behavior to develop a strategy in order to reach complex polypeptide based architectures through bottom-up approaches Preliminary in vitro evaluation in selected cell models in terms of biodegradability, biocompatibility and cellular uptake is presented. Furthermore, after an adequate labeling with fluorescence/NIR probes or/and cation complexing moieties towards the use of MRI and/or PET techniques, the in vivo fate (pharmacokinetics and biodistribution) of our polyglutamates is also described. Preliminary results suggest that they were non-toxic entities, validating them as possible carriers for drug delivery. The covalently entrapped unique architectures have been ultimately used to reach carriers for BBB crossing by means of surface modifications with targeting units and imaging agents. Their BBB crossing properties have being explored in vivo, reaching at least 1.2 % of injected dose in the brain. Thus, those results make them optimal candidates to be used in AD treatment. Among all the biological hallmarks of AD, we are centering our efforts in the amyloid pathway, by the use of curcuminoids and with a neuroprotective approach by combining them with the presence of propagyl moieties within the construct. Their biological output regarding cellular uptake, cell viability, drug release profile and biodistribution has been investigated. Moreover, proof of concept of their activity was achieved in vitro, in organotypic hippocampal cultures and is currently being validated in vivo. Finally, the potential of PGA-based conjugates as tissue-specific smart imaging probes is also explored within the frame of the European consortium LIVIMODE. The combination of NIRF enzyme specific smart probes together with the tissue specificity provided by PGA as carrier is explored to be applied in the early detection of disease-related events in vitro as well as in vivo. This strategy could be used for the development of theranostics towards the early detection and treatment of neurodegenerative disorders
