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Title: Design, sssembly, and biological evaluation of protein nanoparticles as theranostic agents and for photodynamic therapy
Author: Al-Ani, Ali Waleed Numan
ISNI:       0000 0004 6421 2838
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2017
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Nanoparticles derived from proteins offer a smart material for the design of a new generation of anticancer therapies. In this thesis we describe a variety of novel photodynamic therapy (PDT), drug delivery, and imaging agents that have been combined to produce theranostic systems. The initial research focus was to produce a protein nanocage of Listeria innocua DNA binding protein from starved cells (LiDps) presenting the Gaussia princeps luciferase enzyme (GLuc) on its exterior (GLuc-LiDps) together with the Zn (II)-Protoporphyrin IX (ZnPP) photosensitiser that has been covalently attached to the protein surface. This system operates as a PDT based on Bioluminescence Resonance Energy Transfer (BRET). The cytotoxic effect of GLuc-LiDps-ZnPP nanoparticles in the presence of its substrate (coelenterazine) was tested against two types of breast cancer cell lines: SKBR3, MDA-MB-231, and also the MRC5 non-cancerous cell line, by means of an MTT assay. The results indicated that GLuc-LiDps-ZnPP nanoparticles plus coelenterazine could inhibit the growth and the migration of SKBR3 cells, out of those studied. Downregulation of the Bcl-2 and Mcl-1 anti-apoptotic proteins was also observed after treatment with GLuc-LiDps ZnPP-mediated PDT, suggesting the SKBR3 cells may be undergoing apoptosis. Furthermore, both flow cytometry analysis and confocal microscopy images demonstrated that the GLuc-LiDps-ZnPP appeared to be preferentially internalised in SKBR3 and MDA-MB-231 cell lines without uptake in the MRC5 cell line. Reactive Oxygen Species (ROS) levels were significantly increased in SKBR3 cells compared to MDA-MB-231 cells in the presence of this PDT agent. Similarly, a C-terminal mini Singlet Oxygen Generator (miniSOG) photosensitiser was conjugated to LiDps and human apoferritin (HuAft). Moreover HuAft was fused with the ZHER2:342 Affibody (Afb) targeting peptide to form Afb-HuAft. This 6.7 kDa Afb protein has a high binding affinity for the Human Epidermal Growth Factor receptor (HER2), which is overexpressed on the surface of a number of tumour cells especially breast cancers. Both miniSOG-LiDps and miniSOG-HuAft fusion proteins were successfully expressed and purified and their subunits were self-assembled to form GLuc-LiDps:miniSOG-LiDps and Afb-HuAft:miniSOG-HuAft hybrid chimeric cages. Additionally GLuc was directly fused with miniSOG supplemented with LTVSPWY targeting peptide to produce a novel (GLuc-miniSOG-LTVSPWY) for targeting of HER2 overexpressed cancer cells, potentially applicable for PDT. Further study is required in order to thoroughly characterise the GLuc-miniSOG-LTVSPWY and the hybrid cages as well as determine their respective cytotoxicity. In the second objective, lead sulfide quantum dots (PbS QDs) were utilised as a drug delivery system and an imaging agent. The PbS QDs were capped with a cancer cell targeting agent (mutated Afb (Afb2C)) to form Afb2C-PbS QDs. This construct was further modified by conjugation with ZnPP to produce ZnPP-Afb2C-PbS QDs. The cytotoxic effect of Afb2C-PbS and ZnPP-Afb2C-PbS QDs were studied in vitro using SKBR3 (HER2 positive) and MDA-MB-231 (HER2 negative) cell lines. The results indicate that both types of PbS QDs display anti-proliferative activity against SKBR3 cells through inducement of cancer cell apoptosis and/or necrosis. This was observed from Pre-G1 phase arrest and an increase in cell population in late apoptosis and necrosis. These results may contribute to the development of cancer treatment using of nanoparticles derived from protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RS Pharmacy and materia medica