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Al-Ani, Ali Waleed Numan (2017) Design, sssembly, and biological evaluation of protein nanoparticles as theranostic agents and for photodynamic therapy. PhD thesis, University of Nottingham.

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Abstract

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.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Thomas, Neil R. Soultanas, P.
Keywords:	Bioluminescence Resonance Energy Transfer, Gaussia luciferase, Listeria innocua DNA binding protein, photodynamic therapy, zinc (II) protoporphyrin IX, Lead sulfide quantum dots
Subjects:	R Medicine > RS Pharmacy and materia medica
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	45102
Depositing User:	AL-ANI, ALI
Date Deposited:	16 Oct 2017 08:36
Last Modified:	06 May 2020 13:48
URI:	https://eprints.nottingham.ac.uk/id/eprint/45102

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