Nanoparticle theranostics targeting CLEC14A on tumour vasculature

Pearce, Jack (2020). Nanoparticle theranostics targeting CLEC14A on tumour vasculature. University of Birmingham. Ph.D.

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Abstract

This thesis studies the development of a method to knockdown CLEC14A in-vivo. CLEC14A has been shown to have strong pro-angiogenic signalling properties and its knockdown in-vitro has been shown to decrease wound healing and tube formation. Additionally, CLEC14A knockout mice showed a decreased rate of tumour growth in implanted Lewis Lung Carcinoma tumours. siRNA is a common tool used to knockdown genes but needs protection from degrading enzymes found in serum. To this end, a chitosan-based nanoparticle was developed to deliver siRNA. A 60% knockdown of CLEC14A was achieved in-vitro using untargeted chitosan nanoparticles. Microarray analysis of HUVEC cells treated with siRNA entrapped nanoparticles showed that CLEC14A was not knocked down at an mRNA level but that endothelial genes related to blood flow were affected in a similar manner to an increase in laminar blood flow, suggesting that CLEC14A has a regulatory role in endothelial gene expression. Conjugating antibodies to the surface of the chitosan nanoparticles may increase cellular uptake and improve knockdown of CLEC14A. Antibody targeting of the nanoparticles did not improve knockdown, and in fact, decreased efficiency at higher concentrations. Bio-distribution studies were performed with untargeted nanoparticles and showed localisation to tumour vascular endothelium. Nanoparticles were found in the liver and kidneys as well as the tumour.

An anti-angiogenic, CLEC14A based, vaccine was developed to be delivered by the chitosan nanoparticles. A CLEC14A-Tetanus FrC fusion protein was produced in HEK293 cells but failed to fold correctly and be excreted at a sufficient concentration. A CLEC14A-VLRB fusion was created and cloned into a bacterial plasmid. Two fragments of CLEC14A were created to fuse to VLRB with the small fragment being successfully synthesised and purified ready for in-vivo immunisation studies.

Research data from Appendix 5 "Chitosan nanoparticle (CLEC14A siRNA) knockdown in HUVEC: microarray analysis" can be found on the University of Birmingham eData repository at: https://doi.org/10.25500/edata.bham.00000415

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):