Michail, Christos and Allsop, David (2015) Development of peptide inhibitor nanoparticles (PINPs) for treatment of Alzheimer’s Disease. Masters thesis, Lancaster University.
2015ChristosMScbyResearch.pdf - Accepted Version
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Abstract
Purpose: To investigate the best carrier technology for our β-amyloid (Aβ) aggregation inhibitors by developing three types of liposomes (a) plain liposomes, (b) MAL-PEG liposomes, and finally the combination of retro-inverted peptide RI-OR2- TAT (Ac-rGffvlkGrrrrqrrkkrGyc-NH2) attached onto the surface of MAL-PEG liposomes, creating Peptide Inhibitor Nanoparticles (PINPs) of three different sizes (50, 100 and 200 nm). In addition, these nanoliposomes (NLPs) (with particular focus on PINPs) were examined for their ability to affect Aβ aggregation, and to protect against Aβ cytotoxicity. Methods: The creation of NLPs was carried out by the use of a mini extruder, while the elution of PINPs from a size exclusion column was assessed by Dynamic Light Scattering (DLS). The quantification of peptide bound to liposomes was determined by bicinchoninic acid (BCA) assay, while phospholipid content was quantified by Wako phospholipid assay. The effects of the different types of liposomes on Aβ toxicity and viability of SHSY-5Y neuronal cells were examined by MTS assay, whereas effects on Aβ aggregation were determined by Thioflavin-T (Th-T) assay. In addition, a cell penetration assay was carried out in order to examine the ability of liposomes to penetrate into neuroblastoma SHSY-5Y cells. Results: Low concentrations of PINPs 0.1 μM inhibited Aβ aggregation and toxicity in vitro. MAL-PEG liposomes and PINPs were able to penetrate into neuroblastoma SHSY-5Y cells and were also more stable than simple liposomes. Stability means the ability of liposomes to keep their size and their shape stable for long time. In addition, the three types of liposomes were not toxic towards SHSY-5Y neuroblastoma cells. Cytotoxicity is the quality of being toxic to cells. So, none of the three types of our liposomes showed any negative effect on the viability towards SHSY-5Y neuroblastoma cells. Conclusion: NLPs are an ideal carrier for our aggregation inhibitors because they affect Aβ aggregation and toxicity at low doses, and according to other data generated by our group, can cross the Blood Brain Barrier (BBB).