ERL-based nanocomplexes with potential biomedical uses

Abstract

Over the years material science has pursued to mimicking the properties and features inherent to natural proteins. One of these proteins that offer biocompatibility, elasticity and thermosensitive behavior that leads to self-assembled processes is the elastin. From (VPGXG) repeated motifs a variety of Elastin-like recombinamers (ELRs) inspired in natural elastin have been developed. The aim of this thesis is to demonstrate the versatile properties of ELRs in order to create a variety of ELRs able to form nanocomplexes and be applied for biomedical purposes as gene or even drug delivery. The present work has addressed the whole process of design, production, purification, characterization and direct application of the novel ELRs. For that, the utilization of genetic engineering, microbiology, physico-chemical together with in vitro and in vivo techniques has been performed. Initially, the design of ELRs through recombinant techniques and focused on gene delivery applications was achieved. A collection of hydrophilic ELRs rich in lysine and histidine amino acids was obtained. In addition, the incorporation of new functionalities by means of penetratin CPP and LAEL peptides was accomplished. Chemical modifications led to the generation of novel ELRs provided of buffering capacity and cell specificity by means of imidazole groups and anti-MUC1 aptamer incorporations. Indeed, the suitability of a self-assembled ELR diblock (ELRBC) modified with Arg8 CPP at C-t for pH sensitive drug delivery was accomplished. This work explored the cell uptake and accumulation of ELR nanoparticles into acidic vesicles in order to evaluate the incorporation of Arg8 peptide to the ELRBC as the best alternative for carrying the drug. In addition, macropinocytosis as cellular entrance pathway was confirmed by the role of heparin sulfate proteglycan (HSPG) and p21 activated kinase 1 (PAK1) molecules. In addition, the evaluation of ELRs with the biofunctional domains penetratin, LAEL and imidazole groups was assessed in terms of biocompatibility, complexation and transfection abilities. The results showed blood and cell compatibility. Indeed, ELRs were able to condense the plasmid DNA (pDNA) and form polyplexes. These polyplexes were uptaken by cells and they were able to express both p53 and luciferase transgenes showing higher expression for polyplexes formed by LAEL and penetratin peptides. In light of these results, the use of the ELR with high density of lysines was achieved. Higher complexation and transfection abilities were achieved in comparison with the previous ELRs modified with functional domains. Indeed, the incorporation of the anti-MUC1 aptamers in order to provide cell specificity to polyplexes was achieved. As result, stable polyplexes with higher transfection ability in breast cancer cells was obtained. Hence using this system, suicide gene therapy was tested by means of PAP-S gene transfection with high cellular death in vitro. The translation into the in vivo scenario using the PAP-S suicide gene therapy led to inhibition in breast tumor growth evolution. Finally, this work explored the utilization of a complex system constituting a double safety lock device focused on suicide breast cancer gene therapy. The previously designed biopolymer VOK-PEG-5TR1 provided with cell type specificity was shown to form proper polyplexes in presence of plasmid DNA. Thus, the biopolymer complexed with a therapeutic plasmid containing the ricin gene was tested both in vitro and in vivo. High levels of cytotoxicity were found in the target cells while a protective effect was observed over fibroblasts. In vivo assays with different doses of nanocomplexes showed a significant inhibition of tumor growth when compared with placebo.