Biochemical and Microscopy Approaches for Tracking the Delivery of Oligonucleotides

This study explores the effectiveness of gold nanoparticles (AuNPs) and dendritic nanoparticles (NPs) as delivery systems for oligonucleotides (ONs) and small interfering RNAs (siRNAs), respectively. In particular there is a keen focus on the use of microscopy and advance microscopy in order to further elucidate the delivery, which is then further confirmed by biochemical assays. AuNPs demonstrated a potent, non-toxic delivery system for ONs, with increasing cellular uptake observed over time, particularly localising in the perinuclear region. In parallel, amphiphilic dendrimer NPs were investigated for siRNA and ASO delivery, and these dendrimers were shown to be efficient couriers. Although modifications with arginine endings did not preferentially target mitochondria in vivo, as hypothesised, further investigation revealed that they did provide the highest delivery efficiency to isolated mitochondria, which may suggest a potential for targeted delivery. Additionally, larger dendrimers were evaluated for DNA delivery, demonstrating mixed results. It appeared that specific modifications would improve delivery, while other modifications had little to no effect, and further work is required to understand the specific reasons. In order to quantitatively assess subcellular delivery, a novel ‘slot-blot’ method was developed, providing a means to evaluate the mitochondrial localisation. This technique confirmed effective cargo delivery to the mitochondrial matrix, and so may offer a valuable tool for optimising delivery mechanisms. Finally, a novel peptide-based approach for mitochondrial delivery was developed, using constructs derived from the Diablo protein. The MTS construct successfully localised within mitochondria, with potential applications for therapeutic peptide delivery. This system shows promise for targeting mitochondrial processes, such as inhibiting Mia40 to impair mitochondrial DNA repair in tumour cells. In conclusion, both AuNPs and dendritic NPs represent promising vectors for nucleic acid delivery to cells, with specific modifications enhancing their efficiency and targeting capabilities. While we did not come to a clear conclusion of mitochondrial delivery in vivo, a peptide-based delivery mechanism shows promise in this regard. Further optimisation and investigation into these delivery systems could enhance their applications.