Modified folates for selective delivery into cancer cells

Smith, Gareth 2022. Modified folates for selective delivery into cancer cells. PhD Thesis, Cardiff University. Item availability restricted.

Preview	PDF - Accepted Post-Print Version Download (15MB) | Preview
	PDF (Cardiff University Electronic Publication Form) - Supplemental Material Restricted to Repository staff only Download (404kB)

Abstract

Small molecule drugs suffer from prompt resistance and off-site activity. To address these problems, small molecules were substituted with peptides that mediate more sophisticated pathways such as apoptosis through more specific and intricate cellular interactions that require more time to develop resistance. In this research, a dual conjugate system was developed using a luminescent protein such as super folder green fluorescent protein (e.g sfGFP) for highlighting and a pro-apoptotic peptide (e.g. Bid-BH3) for killing. Both were labelled with folate which provided both protein and peptide increased cell target specificity. Folic acid is a high affinity ligand for its dedicated membrane receptor folate receptor alpha (FR-α) which is overexpressed in one third of all cancer. Therefore, folic acid was employed as a protein and peptide delivery motif because the expression pattern of FR-α in healthy cells is largely non-existent. The folic acid labelled sfGFP had preference to interact with FR-α positive cells (e.g. KB cells), and no interaction with FR-α negative HEK293 cells when tested at concentrations below 10 μM. Folate labelled sfGFP was found to interact with KB cells at 10 nM according to FACS. Once this was discovered, sfGFP was swapped for Bid-BH3 for conjugation with folic acid and this was compared alongside its unmodified sequence. It was found that the folate labelled sequence retained its cytotoxicity when compared to the native sequence on KB cells in the absence of free folic acid (e.g. ± 0.5, 5.3 μM vs. ± 1.0, 6.2 μM respectively). In the presence of free folic acid cytotoxicity of the folate labelled peptide diminished, whereas the native sequence cytotoxicity was unaffected. This result was also observed when both sequences were tested on FR-α-negative HEK293 cells. In other works, the synthesis cell penetrating peptides (CPP) based on TAT and aurein-1.2 connected to folic acid was attempted. These sequences were planned for conjugation to sfGFP to generate a protein-based conjugate with a combination of cell targeting and endosome escape. The plan was to swap sfGFP for a cytotoxic protein once ideal concentrations allowing for cell targeting and endosome escape were found. Many issues were found when synthesising the TAT sequences such as glutarimide formation, truncation and failed folic acid labelling. The aurein-1.2 sequences were significantly easier to synthesise, however, folate labelling was problematic, and the peptide sequence was found to rapidly self-cleave from sfGFP in phosphate buffered saline. Because of these results, alternative synthetic techniques for producing folate-CPP conjugated to sfGFP will need to be considered.

Item Type:	Thesis (PhD)
Date Type:	Submission
Status:	Unpublished
Schools:	Chemistry
Date of First Compliant Deposit:	28 April 2023
Last Modified:	28 Apr 2024 01:30
URI:	https://orca.cardiff.ac.uk/id/eprint/159092

Actions (repository staff only)

Edit Item