Advanced nanodevices based on elastin-like recombinamers as smart drug delivery systems for biomedical applications

Resumen

Despite of the numerous advances in biomedicine, there are some issues to be resolved. One of these limitations is the low specificity of therapeutic agents with undesired side effects on healthy tissues. Thus, novel strategies for achieving an accurate action on targeted cells are needed. Over the years, advanced biomaterials have emerged as one of the most promising tools to increase therapeutic efficiency and biocompatibility, and possess the ability to mimic properties and features found in natural macromolecules in order to use in multiple biomedical applications, such as tissue engineering or drug delivery. Among the novel biomaterials, we can find Elastin-Like Recombinamers (ELRs). ELRs are protein-based polymers composed by the repetition of VPGXG (Val-Pro-Gly-X-Gly) pentapeptide, where X can be any amino acid except proline. This pentapeptide can be found in the sequence of natural elastin protein, a major component of extracellular matrix (ECM) and confers some characteristic features to ELRs. Thus, ELR-based biomaterials show biocompatibility, biodegradability and stimuli-responsive behavior. Moreover, ELRs are defined by the Inverse Temperature Transition (ITT), a reversible phase transition above the so-called Transition temperature (Tt). When the Tt is reached, ELRs change from a disordered soluble state to self-assemble into well-defined structures, from the macro to the nanoscale, such as nanoparticles, micelles or vesicles. Due to the recombinant origin of ELRs, the Tt of these biopolymers can be modulated depending on the polarity of side chains of amino acid placed in the guest position. Thus, the molecular design allows us to obtain in the lab different ELRs with self-assembling ability in response to characteristic Tt and achieve smart devices for biomedical applications. This Thesis describes novel ELR-based biomaterials as a promising approach for biomedical applications and their potential use as therapeutic tools for different diseases. A novel strategy for DNA vaccination against a viral antigen was developed, thereby obtaining a fusion gene library with different properties and behaviour under physiological conditions, and several nanodevices derived from multiple ELR constructs were tested. Natural antigens usually trigger low immunogenic responses, so novel strategies for modulated immunogenicity are needed in order to achieve safer vaccines. By taking advantage of genetic engineering techniques, ELR genes with different hydrophilic/hydrophobic nature were fused to the gene codifying for Gn glycoprotein from Rift Valley fever virus (RVFV). Eukaryotic cells were transfected with DNA constructs, cell viability was measured and protein expression was evaluated by immunocytochemistry and confocal microscopy. At light of results, gene constructs whose ELR block involved the presence of hydrophobic amino acid, as valine, improved the expression of Gn glycoprotein. Also, DNA vaccination was performed in an in vivo mouse model and immune response to viral antigen was studied. Results determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge and were better inducers of cellular immunity, as judged by in vitro re-stimulation experiments. Thus, results highlighted the potential of ELR biomaterials for DNA vaccination strategies against Rift Valley fever virus infections and achieve higher yield productions of natural antigens. The second chapter of this work is focused on the description of the design and development of novel ELR polymers based on an amphiphilic backbone with multiple bioactive sequences in order to selectively release a therapeutic peptide thereby modulating the cell growth. One of the most important problems of current medicine is the lack of selective agents which only affect targeted cells. Chemotherapeutic drugs are such an example, as this kind of agents tackles both tumor and healthy tissues. By means of genetic engineering techniques, two complex biopolymers were developed involving multiple blocks in order to achieve full control of the release of a therapeutic peptide in the cellular cytoplasm. Thus, internalization pathway, intracellular fate and spatiotemporal action were driven by the molecular complexity of ELR biopolymers. Physicochemical characterization demonstrated that these two biopolymers were able to self-assemble into nanoparticles with adequate size and surface charge for intracellular delivery of therapeutic peptides. The internalization pathway and intracellular activation were also determined. Finally, the cytotoxic effect of nanoparticles on cancer cells and non-cancer cells was compared and results showed an enhanced effect over cancer cells with minimum toxicity in non-cancer ones. Thus, results showed that a smart complexity was successfully acquired to achieve the controlled delivery of a therapeutic peptide and avoid aberrant proliferation of targeted cells. The following chapter describes the potential use of these self-assembling ELR-based nanoparticles carrying a peptide for the inhibition of Akt phosphorylation as therapeutic nanodevices against pancreatic cancer. By taking advantage of patient-derived cells, the developed nanoparticles were tested in a closer model to real disease and heterogenic tumor populations. First, the in vitro assays showed that developed nanocarriers possessed a time and dose-dependent effect not only in pancreatic cancer cells metabolism, but also in cell viability. Flow cytometry and confocal microscopy assays were performed in order to determine cell uptake and intracellular localization. At light of the results, ELR-based nanoparticles co-localized with lysosomes in the cellular cytoplasm. Synergic effect of developed nanodevices and light-derived therapy was also studied. Thus, combination of ELR nanoparticles and photosensitizer TPP2Sa appeared to have synergic effect and significantly affected pancreatic cancer cell metabolism. Furthermore, a mouse animal model was used in order to determine the pharmacokinetic profile and biodistribution of nanocarriers in vivo. At light of the results, ELR-based nanoparticles met the requirements for drug delivery systems. Finally, Chapter 4 is focused on the development of novel hybrid nanodevices as advanced drug delivery systems with combined therapeutic approaches for colorectal cancer treatment. For this purpose, a dual hybrid ELR nanodevice was designed as a consequence of the combination of two previously described amphiphilic polymers: one forming highly monodisperse nanoparticles with encapsulated chemotherapeutic agent Docetaxel and another one consisting on smart nanoparticles carrying an Akt inhibitor previously described in Chapter 2. Thus, the resulting device was characterized and in vitro efficacy was tested on cancer and endothelial cells. Results from the in vitro assays showed that targeted nanohybrids selectively affect cancer cells by two different ways: Akt inhibitor triggered early apoptosis whereas DTX elicited both apoptosis and necrosis at longer times. A reliable colorectal cancer animal model close to real disease was produced in order to study the anti-tumor effect of systemically injected ELR nanohybrids. Magnetic Resonance Imaging showed that tumor-associated inflammation was decreased after intravenous administration of the ELR-based nanodevices. Ex vivo analysis showed that dual-approach ELR nanohybrids significantly decreased the number of tumor polyps, whose size was also reduced. Moreover, histological analysis determined that those animals treated with ELR nanohybrids possessed improved tissue architecture and crypt morphology in the distal colon. In summary, this Thesis explores new insights about nanodevices for biomedical applications involving multiple diseases. In this work, all the necessary steps needed for such aim are included, from gene design and cloning, bioproduction in a prokaryotic organism, purification, synthesis and physicochemical characterization to the assessment of both in vitro and in vivo behavior.