AbstractThe RALA peptide delivery system complexes anionic compounds through electrostatic interactions, to form a nanoparticle with the physiochemical characteristics to cross cellular membranes and escape endosomes; thereby releasing the cargo intracellularly to exert the optimal therapeutic effect.
The focus of this thesis was to repurpose the nitrogen-containing bisphosphonate (N BP), alendronate (ALN), into a novel anticancer therapeutic through complexation with the RALA peptide. N-BPs inhibit the prenylation of GTPase proteins, which are responsible for cell replication, morphology and signalling, to cause cell death through apoptosis. However, the in vivo therapeutic use of bisphosphonates is limited due to low bioavailability and adverse effects such as necrosis of the jaw. When administered intravenously, free bisphosphonates preferentially bind to the bone microenvironment with only negligible internalisation in extraskeletal organs.
Multiple RALA variant peptides were designed to investigate the role of histidine in the sequence with the aim of reducing the sequence length without compromising functionality. RALA26 produced transfection efficiencies comparable to the RALA peptide at N:P ratio 6 with comparable alpha-helicity. RALA26 therefore holds promise as a functional truncated RALA-derived peptide and warrants further investigation.
The anticancer effects of ALN were investigated through a series of in vitro studies in PC-3, LN229 and T98G cell lines. Dose response studies demonstrated dose and time dependent cytotoxicity with induced cell apoptosis when treated with RALA/ALN nanoparticles. The mechanism of N-BP action was also studied which revealed RALA/ALN nanoparticles inhibited the prenylation of H-Ras, thus the GTPase protein was unable to bind to the cell membrane compared to untreated or ALN only controls. Furthermore, RALA/ALN nanoparticles remained stable post-lyophilisation without compromising on functionality in vitro.
RALA++, a MMP-2/MMP-9 cleavable PEG moiety, was employed in vivo to enhance tumour targeted specificity and to avoid clearance of RALA/ALN nanoparticles via the mononuclear phagocyte system. Lyophilised and fresh RALA/ALN and RALA++/ALN nanoparticles were evaluated in PC-3 xenograft tumour models. Fresh and lyophilised RALA/ALN nanoparticle formulations significantly decreased tumour growth following intravenous administration with survival at 56 and 70 days, respectively, compared to lyophilised RALA++/ALN nanoparticles (49 days) and untreated controls (31 days). In contrast, mice in which treatments were delivered locally, displayed increased survival with lyophilised RALA++/ALN nanoparticles compared to RALA/ALN particles. Future studies should investigate the biodistribution of the formulated nanoparticles to elucidate the dose per tissue and pharmacokinetics so that the optimal therapeutic index can be found.
|Date of Award||Jul 2020|
|Supervisor||Helen McCarthy (Supervisor), Thakur Raghu Raj Singh (Supervisor) & Cian McCrudden (Supervisor)|