Development and application of polymeric nanoparticles for intra-articular delivery of mRNA

Abstract

Osteoarthritis (OA) is a progressive and degenerative disease of the joints affecting over 500 million people worldwide, characterized by inflammation and loss of the cartilage. The aim of this thesis is to present the development and application of a new class of bioreducible poly(amidoamine)-based nanoparticles as a non-viral vector for local mRNA delivery in the joints. These polymeric nanoparticles (PNPs) were tested and optimized in different in vitro, ex vivo, and in vivo models for delivery of mRNA-encoding reporter genes, showing their potential for future application in OA treatment. Chapter 2 presents a systematic review about the state of the art of natural and synthetic PNPs for drug delivery in OA research. In Chapter 3, we show the design of a novel multi-functionalized, branched poly(amidoamine) (PAA) possessing bioreducible disulfide bonds, which can be easily formulated with mRNA to form PNPs in an aqueous buffer. The inclusion of quinoline (Q) side chains increased the mRNA-polymer stability and transfection efficiency in COS-7 cells, and the co-formulation with a PEG-polymer coating produced smaller, electroneutral PNPs with improved stability. These nanoparticles with both Q and PEG-polymer coating induced significantly higher luciferase activity in mice muscle than uncoated NPs, following intramuscular injection of PNPs loaded with luciferase mRNA. In Chapter 4, we tested these bioreducible PAA-based NPs (loaded with EGFP mRNA) in the C28/I2 human chondrocyte cell line, for confocal imaging of their intracellular trafficking and evaluation of transfection efficiency. Cell uptake was higher and faster for the positively charged uncoated NPs, compared with the neutral PEG-coated NPs, but transfection efficiency was lower. Furthermore, endosomal entrapment of these PNPs decreased over time, and mRNA release could be visualized both in vitro (by agarose gel) and in live cells (by confocal microscopy). Transfection efficiency was maximized using a systematic Design of Experiments (DoE) approach to nearly 80% of GFP-positive cells and without toxic effects. In Chapter 5, we investigated the intra-articular (IA) application of these PAA-based NPs for local mRNA delivery in the joints. To this end, uncoated and PEG-coated NPs were formulated to test mRNA delivery in more complex joint models: (1) a 2D culture of C28/I2 chondrocytes supplemented with synovial fluid, and (2) an ex vivo culture of mouse knee joints following IA injection of the PNPs. While the neutral PEG-coated NPs had a smaller particle size and higher cell uptake in the presence of synovial fluid (in vitro), the positively charged uncoated NPs appeared to display higher cartilage penetration and uptake in ex vivo culture of mouse knee joints. These results suggest that, apart from nanoparticle size, electrostatic interactions between cationic PNPs and the anionic components of the extracellular matrix (ECM) play a key role in cartilage penetration and mRNA delivery to tissue-resident chondrocytes. Altogether, we demonstrated how crucial it is to validate novel delivery systems in models mimicking the joint milieu (e.g., synovial fluid and a dense ECM). As a conclusion, these PNPs are a promising alternative for intra-articular delivery of mRNA for therapeutic applications in OA.