Abstract
Stroke is the second leading cause of death and the third leading cause of disability worldwide. Ischaemic strokes account for 85% of all cases and are thrombotic in nature. Although restoring blood flow quickly is critical, the only approved non-surgical treatment involves the use of clot-dissolving agents such as recombinant tissue plasminogen activator (r-tPA). In clinical practice, however, only 1-5% of stroke patients receive r-tPA due to its narrow therapeutic window of 3 hours and the increased risk of intracranial haemorrhage. Thus, most stroke patients rely on neuroprotective treatments to preserve the salvable penumbra region before it progresses to irreversible ischaemic tissue. Nose-to-brain delivery enables a rapid onset of action for neuroprotective therapies, allowing them to bypass the blood-brain barrier (BBB) and reach the brain within minutes. This approach can potentially be administered by paramedics before the patient is transported to the hospital. However, therapies need to be enzymatically stable, selectively target the receptor of interest, permeate the BBB, and degrade into non-toxic metabolites.This thesis aimed to engineer an enzymatically stable and brain-permeable Angiotensin-(1- 7) peptide [H-Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-OH (A1-7)], which has been shown to be neuroprotective in ischaemic stroke by exhibiting vasodilatory, anti-inflammatory, anti- oxidative, and angiogenic activities via binding to the orphan Mas G-protein coupled receptor (MasR). A1-7 was modified at the Tyrosine4 by palmitoylation, using a method patented by Dr. Lalatsa, to induce the peptide to self-assembly into stable nanostructures in aqueous environments. Peptides were characterised for their primary structure and self-assembly and for their in vitro stability in plasma, brain, and liver homogenates. A1-7 and its stable analogues were loaded into nano-in-microparticles of biodegradable quaternised glycol chitosan (QGC) or quaternised pullulan (QPUL) to enhance particle residence time in the nasal mucosa, target the olfactory bulb and allow enzymatic stability and permeability. Binding studies of A1-7 and analogues were performed in silico using a MasR Alphafold model equilibrated into a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) membrane for 1,000 ns. Binding studies of peptides were performed by a direct peptide-MasR interaction assay using murine membrane protein fraction and Texas red-labelled fluorescent peptides. Signal transduction was also assessed in terms of cyclic AMP (cAMP) production and intracellular calcium release, by using human RPMI 2650 cell line expressing MasR and a model of murine optic nerves developed by Prof Butt, respectively. Toxicity studies of the peptides were conducted in human BBB cells (SC-1800, HBVP, hCMEC/D3), murine primary brain cells (astrocytes, pericytes, microglia), red blood cells (RBCs), and human nasal cells (RPMI 2650), with the latter also including assessments of nano-in- microparticles toxicity. MasR expression in both murine brain cells and tissues, as well as in human cell lines, was assessed using immunochemistry and RT-qPCR. Neuroprotective assays were conducted in: (i) primary brain cells under oxidative stress (1-h peptide pre- treatment followed by 4 h of 200 µM H2O2) and assessed using cell metabolic activity assays (MTT and Resazurin) and oxidative probes (Dihydroethidium, DHE); (ii) organotypic brain slice cultures under oxidative stress (1-h peptide pre-treatment followed by 4 or 24 h of 2.5 mM H2O2) through the quantification of oxidative and inflammatory genes by RT-qPCR. Additionally, peptide permeability was assessed using a Texas-red labelled lipidised A1-7 peptide (TRTA1-7) administered in vivo in mice via nose-to-brain route, and peptide biodistribution was evaluated in different organs at various time points (5 min, 10 min, 60 min, 240 min, 1440 min) using both HPLC and histochemistry.
Lipidised peptides were successfully synthesised and characterised, forming stable twisted nanofibers with widths of 10-30 nm. They demonstrated significantly higher stability compared to the parental peptide in all homogenates tested, with lipidised A1-7, TA1-7, exhibiting a 68-fold, 55-fold and 27.7-fold half-life increase in plasma, brain and liver homogenates. Nano-in-micro particles were successfully synthesised, exhibiting a corrugated morphology that likely promotes effective aerosolization and a size range of 8- 12 µm, which favours drug deposition in the upper respiratory tract. In silico and in vitro binding studies have confirmed a higher affinity of the lipidised peptide for the MasR receptor, attributed to the more stable β-sheet structure of the peptide and the insertion of the lipidic tail within the membrane. Binding resulted in a reduction of cAMP levels across all peptides, while only the lipidised peptide caused a significant decrease in intracellular calcium storage compared to A1-7, which did not exhibit any calcium response. Peptides didn’t show any toxic effect in all cell types up to at least 25-50 µM. For the lipidised peptides, toxicity was concentration-dependent. Haemolytic studies indicated the absence of toxic effects for all peptides at concentrations up to at least 80 µM. MasR was detected in all cell lineages tested, with the highest expression in astrocytes and neurons, as well as in the hippocampus (CA1, dentate gyrus), piriform cortex, and hypothalamus. Neuroprotective studies in primary brain cells under oxidative stress demonstrated significant increases in cell metabolic activity for TA1-7 and A1-7 in microglia and astrocytes, while the effect in oligodendrocyte progenitor cells (OPCs) was not significant. In organotypic brain slices, after 4 h of oxidative stress, TA1-7 and A1-7 predominantly exhibited high NOS2 and NOS3 expression. However, after 24 h, inflammatory effects were observed due to the high expression of IL-6 (only associated with TA1-7) and PTGS2. Finally, TRTA1-7 was found at a high concentration in the brain (47.97 ± 7.89 µg g-1) 5 min after nose-to-brain administration and maintained a consistently high concentration for at least 24 h (36.37 ± 2.86 µg g-1), indicating its potential as an excellent candidate for nose-to-brain delivery of A1-7 peptide analogues. Thus, overall these data demonstrate that the peptide exhibits enhanced enzymatic stability and brain permeability, enabling rapid nose-to-brain administration for potential applications in neuroprotection, such as post-ischemic stroke treatment.
Date of Award | 11 Jan 2025 |
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Original language | English |
Awarding Institution |
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Supervisor | Arthur Butt (Supervisor), Aikaterini Lalatsa (Supervisor) & Andrea Bucchi (Supervisor) |