Well-defined polypeptide-based systems as non-viral vectors for cytosolic delivery

Resumen

A convenient cytosolic drug delivery constitutes a very powerful tool for the treatment and/or prevention of several relevant human diseases. Along with recent advances in therapeutic technologies based on biomacromolecules (e.g. oligonucleotides or proteins),we also require the development of technologies which improve the transport of therapeutic molecules to the cell of choice. This has led to the emergence of a variety of promising methods overthe last 20 years. Despite significant progress, these methods still suffer fromseveral shortcomings including low/variable delivery efficiency, high cytotoxicity, and perhaps most importantly, ineffective endosomal/lysosomal escape. In this context, Polymer Therapeutics (PT) have emerged as anexcitingalternative to overcome suchlimitations. Specifically, well defined polypeptide-based therapeutics could be consideredexcellent candidates for drug delivery due to their suitablebiodegradability,versatility,multivalence and high drug loading capacity. On the other hand, a comprehensive understanding of therapeutic moleculesisalso required for therational selection and design ofan appropriate intracellular delivery carrier. The application of new, robust, and sophisticated characterisation techniqueshas complemented existing techniques to meet the challenge of working under physiological or near-physiological conditions. The remarkable development in the design of drug delivery systemshas forced the establishment of design guidelines to achieve the specific therapeutic effect. The importance of an exhaustive physicochemical characterisation has given rise to more efficient therapeutic strategies via better controlofthe pharmacokinetics and biodistribution of such nanomedicines. On this basis, the main aim of this thesis is focused on two main topics: (i) the design, development,and validation of nanosized polypeptide-based carriers capable of facilitating the cytosolic transport of bioactive agents which are not able to cross biological membranes by themselves or exhibit low lysosomal stability, such as plasmid DNA (pDNA), small interfering RNA (siRNA), orproteins, and(ii) the exhaustive physicochemical characterisation of polymeric drug delivery systems to determine their solution conformation and its correlation with their therapeutic output. In order to accomplish the above-mentioned goals and based on well-established properties of poly-L-glutamic acid (PGA) as a polymer carrier, firstly, we synthesised and evaluated different conjugates ofPGA with succinyl tetraethylene pentamine (Stp) oligoaminoamides as non-viral carriers for pDNA or siRNA delivery. We hypothesised that these zwitterionic bioresponsive and biodegradable carriers may achieve similar transfection efficiencies as those achieved for analogous polycations, but with greater safety in biological scenarios by avoiding polycation-triggered side effects. After physicochemical characterisation of the obtained conjugates, we evaluated cytotoxicity in both N2a and 4T1 cell lines to assesscell viability. We also performed transfection and cell internalisation assays to assess conjugate functionality. We also continuedexploring different alternatives within the field of polypeptides. We synthesised and characterised multifunctional polymeric platforms based on natural or synthetic polyaminoacids, such as PGA, poly-L-arginine (PArg), poly-L-ornithine (P(Orn)), and their derivatives to find an encouraging vehicle for effective siRNA delivery as anticancer treatment. We obtained several oligonucleotide conjugates and complexes and performed preliminary in vitro studies in B16F10-luc-G5 cell line. Upon comparing the obtained results of silencing, we established that P(Orn)-based systems offered the most promising results. Additionally, the feasibility of the delivery of a protein, alanine:glyoxylate aminotransferase (AGT), in order to promote enzyme-replacement therapy in a rare disease Hyperoxaluria Type I by conjugating the enzyme with a polyethyleneglycol (PEG)-PGA block-co-polymer based nanocarrier was also evaluated. Thisconjugation strategy does not significantly alter the functional properties of AGT and endows the protein with the ability to cross the plasma membrane and localise in the cytosol of a cellular model of PH1. Engineering AGT by the insertion of a stronger peroxisomal targeting sequence(PTS)and the mutation of one of the polymer anchoring points located on the “extended PTS1” partly decreases the conjugation efficiency. However, this allows peroxisomal targeting of the conjugates, resulting in the enhanced ability to detoxify intraperoxisomal glyoxylate with respect to the wild-type protein. We obtained all mentioned systems from polymers with verylow polydispersity (Ɖ~1.2) and, therefore, precise and well-defined structures, so allowing reproducibility and the determination of a clear structure-activity relationship (SAR). We also exhaustively investigatedphysicochemical properties of all obtained polypeptides in terms of size and solution conformation. Finally, in order to deeply understand the importance of solution conformation in conjugate therapeutic output, highly advanced physicochemical techniques such as small angle neutron scattering (SANS) or small angle X-ray scattering (SAXS) were used always trying to mimic physiological conditions. Arigorous and detailed structural investigation of polymeric nanosystems already classified as successful drug delivery nanocarriers were performed.