Modulation of Drug Efflux at the Blood-Brain Barrier Through Targeted siRNA Delivery via Nanoparticles

• Main Author: Gomes, Maria João Bidarra Tavares
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2017
• Subjects: Physics
• ISBN: 1083488732; 9781083488732

Central nervous system (CNS) disorders are increasing over the last years as a consequence of a continuously aged population growth. Despite scientific advances, current therapeutics are often not thrived, which raises the increasing need for successful ways to reach CNS and achieve fruitful treatments. Additionally, the blood-brain barrier (BBB) is a unique membrane involving the brain, able to create such restrict CNS environment. Therefore, regarding CNS disorders incidence, failing therapeutics and BBB-related responsibility, the development of in vitro BBB models that mimic the physiologic BBB is a key factor for the study of newly developed drug/gene delivery systems, namely their interaction with BBB and permeability. Simultaneously, the production of effective platforms to circumvent BBB protective functions is leading this research field.Drug efflux pumps at the BBB interface, as P-glycoprotein (P-gp), act as a noteworthy barrier that prevents the effectiveness of CNS disorders treatments, due to their ability to strongly limit the perfusion of compounds into the brain. Over the past decades, new approaches towards overcoming the BBB and its efflux transporters have been proposed, with limited degree of success. Small interfering RNA (siRNA) has been taking place in this topic as consequence of its targeting competence to specifically silence a protein of interest in a post-transcriptional way. Therefore, siRNA is used as a promising approach to selectively silent target proteins, improving the ability of drugs to reach the brain.As important as the efficient protein silence is the transport of siRNA to its anatomical site of action. Nanotechnology and bioengineering joined to carry siRNA to the desired location, protecting the oligonucleotide circulation, directing its transport, and promoting intracellular delivery. Recent research on functionalization strategies offers distinct chemical tools to bind specific ligands to the surface of nanosystems, enabling them to obtain targeted functions. The selection of proper ligands is promoting the BBB surpassing likelihood of siRNA-loaded systems.Gathering these previous ideas, the main aim of this thesis was to develop targeted and safe nanosystems, able to modulate the drug efflux at the BBB via siRNA against P-gp. The first part of this approach was focused on the development of a non-cellular based BBB model where permeability experiments could be performed in a simple and fast way, obtaining a high throughput screening tool, also used for in vivo permeability prediction. Phospholipid vesicle-based permeability assay (PVPA) method was used to produce such model, and then, to assess passive transcellular-like permeability of siRNA, free and loaded into nanoparticles. Both polymeric poly(lactic-co-glycolic acid) (PLGA) and lipid (solid lipid nanoparticles, SLN) nanoparticles encapsulating siRNA were developed. Slightly negative (- 10 mV) and monodisperse populations around 140 nm were obtained and their effect on siRNA permeability through the BBB-PVPA model was assessed. The permeability of siRNA has increased from 3.7  10-6 cm s-1 (free siRNA) to 5.5  10-6 cm s-1 and 6.9  10-6 cm s-1 after encapsulation into polymeric and lipid nanoparticles, respectively.Then, the nanoparticulate-based systems were improved through the assessment of several materials and functionalization techniques. A PLGA-based polymer and a lipid, both amine terminated, were added to the previously formulation systems in order to improve their surface functionalization through carbodiimide and maleimide chemical approaches. Concurrently, the surfactant previously used, poly(vinyl alcohol), was replaced for Tween80, as this last one was easier to remove from nanoparticles surface, enhancing the functional groups availability and therefore exhibiting their mainly negative charge. Therefore, polymeric PLGA nanoparticles presented mean size around 115 nm, negatively charged surface (-30 mV) and 50% of siRNA association efficiency, while SLN, also negatively charged (up to -21 mV), presented 150 nm of mean size and association efficiency up to 52%. The release profile of siRNA from nanoparticles was sustained, reaching around 60% of released siRNA after 24h in physiological conditions. As well, the safety of nanoparticles was demonstrated assessing the metabolic activity of brain endothelial cells, found to be generally above 80%, up to 24h of incubation. To enhance transcellular permeability, nanoparticles were functionalized on the surface with a peptide-binding transferrin receptor (TfR), in a siteoriented manner, obtaining mean sizes between 321 and 506 nm and surface charge within the range -10 to -40 mV. Such modification was confirmed by 1H nuclear magnetic resonance ( 1H NMR), and their targeting ability against human brain endothelial cells was demonstrated by fluorescence microscopy and flow cytometry. The interaction of TfR-targeted nanoparticles with brain endothelial cells increased 3- and 4-fold compared to non-modified SLN and PLGA nanoparticles, respectively.While the interaction with endothelial cells of functionalized and non-functionalized PLGA nanoparticles were evident and detected through fluorescence microscopy, minor differences were found between functionalized and non-functionalized SLN. These data may indicate an inefficient SLN functionalization, either due to a low yield binding chemistry process itself, or due to the lack of correct peptide availability for its receptor. Therefore, polymeric systems seemed to be more benefic that lipid ones, which justifies the selection of PLGA-based nanoparticles for the next project step.Finally, the functionality of TfR-targeted PLGA nanoparticles via different peptide linkage bridges (carbodiimide and maleimide) was assessed on a human BBB cell-based model. Beyond their ability to improve siRNA permeability through the BBB by 2-fold, it was shown that, 96h post transfection, TfR-peptide functionalized PLGA nanoparticles via maleimide chemistry successfully induced reduction of P-gp messenger RNA (mRNA) expression up to 52%, compared to non-functionalized systems. Subsequently, the permeability of rhodamine 123, as a P-gp substrate, through the human BBB model, was determined upon the treatment of endothelial cells with siRNA-loaded TfR-peptide functionalized PLGA systems, resulting in an increase up to 27% in permeability in three hours of assay. This suggested a positive and marked biologic effect of the siRNA-induced P-gp down-regulation.Overall, a BBB-targeted polymeric nanosystem was developed for delivery of siRNA against P-gp. Functionalized siRNA-loaded PLGA nanoparticles showed to be successful in silencing P-gp as a BBB efflux transporter and, consequently, in enhancing the blood-to-brain in vitro permeability of a P-gp substrate. Hence, drug efflux modulation at the BBB level was attained, bringing hope to CNS disorders treatments, since drugs could reach brain in higher and therapeutic concentrations. Additionally, being P-gp commonly over expressed at tumor cells, this polymeric system has the potential to be applied to cancer when properly functionalized to those cells.