Encapsulation of hydrophobic drugs with elastin-like recombinamers by supercritical antisolvent process for advanced biomedical applications

Resum

Nowadays science tends to form multidisciplinary teams, where experts in different areas converge to achieve the same objective. This thesis is one of those examples where knowledge from areas such as genetic engineering, molecular biology, biotechnology, chemistry and chemical engineering is gathered together to achieve a small step forward in the world of science. The development of advanced materials that improve the features of devices already on the market is a general challenge in all technological areas. The emergence of new therapeutic molecules and their validation stimulates the progress of new biomaterials, either as excipients or by exerting an active effect on the dosage and efficacy of the drug. Among the most frequently promoted are those of natural origin: polysaccharides and proteins. The first ones have excellent biocompatibility and capacity to protect the active ingredients but lack the flexibility and multifunctionality of proteins. Proteins perform numerous functions in living organisms and viruses, with great efficiency and selectivity, interacting specifically with other biomolecules and adapting to different physiological environments. Currently, Elastin Like Recombinamers (ELRs) are biopolymers that form unique molecules of completely defined structure with the strength, self-assembly capacity and natural mechanical properties of human elastin. But also with the unparalleled versatility of proteins, where all 20 amino acids can be used in infinite combinations to complement the functionalities not present in elastin. ELR polymers are polypeptides whose sequence consists of repeats of the (VPGXG)n sequence, or permutations thereof, conferring unique mechanical properties similar to natural elastin. They are currently produced by recombinant DNA technology using Escherichia coli bacteria and their properties include some of the most remarkable features of elastin: extraordinary elasticity, self-assembly capacity, and excellent resistance to stress. Controlled drug delivery systems from biodegradable polymer nanoparticles are having a major medical impact in a wide range of therapeutic areas. However, difficulties remain in formulating protein-based nanoparticle systems. For example, there are difficulties in the encapsulation in water-based hydrogels, resulting in very low loadings of this type of drugs, in the exposure of protein polymers to high temperatures, leading to their denaturation, or in the use of organic solvents, in some cases toxic, which implies the design of a subsequent process for their elimination. For these reasons, new production processes have been developed to protect the functionality of these polymers. The use of supercritical fluids (SCFs) is a viable option to consider when producing nanoparticulate systems for controlled drug release. High-pressure technologies, especially those involving supercritical carbon dioxide (scCO2), were studied as sustainable alternatives for polymer processing because of the moderate pressure and temperature at which the supercritical state is reached (73.8 bar and 304.1K) allow the processing of polymers at lower temperatures. The first chapter is focused on acetazolamide, an active compound used in the treatment of glaucoma that is considered the second most common cause of blindness in the world after cataracts and one of the main causes of permanent blindness. in this case, an ELR has been used, which consists of a tetrablock design involving two hydrophilic blocks containing glutamic acid (E) and two hydrophobic blocks containing isoleucine (I) as guest residues. Acetazolamide (AZM) is encapsulated by SAS using DMSO as a solvent in three different ELR:AZM ratios achieving high a process yield, up to 62%. The powder obtained after SAS technique was characterized by SEM and chemically characterized by NMR H+ obtaining a residual amount of DMSO between 6.6 and 12.3%. After dispersion under physiological conditions, the microparticles disintegrate into stable monodisperse nanoparticles due to low PDI (between 0.132 to 0.155) and high Z-potential (between -33 to -34mV). Topographies were confirmed by TEM and by a novel AFM characterization. The in vitro release profiles show a Fick diffusion-governed release with a more sustained release of the drug over time. Transcorneal permeation results show a higher permeation level than the control solution, reaching significant differences of more than 30% after 2h. In vivo intraocular pressure (IOP) tests performed on hypertensive rabbits show higher effectiveness for the three formulations analyzed, suggesting a higher bioavailability of the drug in the treatment. Finally, ocular irritation tests did not reveal any ocular alteration or damage. In the second chapter of this work, a coaxial nozzle was designed, which, combined with the implementation of a bypass line in the installation, allowed us to reduce the residual amount of solvent in the final product. The chemotherapeutic agent Docetaxel (DTX) is encapsulated with a block copolymer ELR containing the RGD peptide, a specific target sequence for cancer cells. the amount of residual DMSO found in the final product after implementing the process improvements was found to be between 3.5 and 3.7%. Physical characterization of the powder obtained after SAS process shows spherical microparticles by SEM and after dispersion under physiological conditions, microparticles disaggregate into stable monodisperse nanoparticles. This protects the drug, as confirmed by NMR analysis, thereby increasing the water solubility of DTX up to fifty orders of magnitude. The delivery process is governed by the Fick diffusion mechanism and indicates that the presence of DTX on the surface of the particles is practically negligible. Cellular assays showed that, due to the presence of the cancer target sequence RGD, breast cancer cells were more affected than human endothelial cells, thus meaning that the strategy developed in this work opens the way to new controlled release systems more precise than non-selective chemotherapeutic drugs. The last chapter covers the treatment of dry eye disease using a combination of two active compounds, resveratrol and quercetin. In this part of the work, the use of DMSO for encapsulation with ELRs was avoided and an ethanol/water mixture was used, for which new operating conditions had to be investigated, based on the CO2-Ethanol-Water ternary diagram. A final product was obtained with different ratios that were physically and chemically characterised. In addition, stability tests showed that the encapsulated active principles display a prolonged degradation over time.