AbstractPancreatic cancer sufferers are faced with a dire prognosis in part because no curative chemotherapy regimen exists. The first-line pancreatic cancer combination therapy of folinic acid, 5-fluorouracil, irinotecan and oxaliplatin (FOLFIRINOX) comes with significant toxicity and is only tolerable by some patients. Some improvement of the therapeutic index of existing chemotherapies may be achieved by encapsulation of chemotherapies inside nanoparticles (NPs) which have distinct physicochemical characteristics in comparison to free drugs. This enables a host of therapeutically relevant benefits such as avoidance of poorly tolerated solvents (e.g. Cremophor-EL in the case of paclitaxel), overcoming cellular drug efflux, improved pharmacokinetics and exploitation of the enhanced permeability and retention effect. Through these benefits several NPs are approved for use against pancreatic and other cancers. Despite this, there is great scope for improvement of their therapeutic properties in the second generation of nanomedicines.
One area of interest is surface modification of the NPs with ligands of specific affinity to improve tumour specific localisation/internalisation and/or produce a therapeutically beneficial effect at the tumour. This concept is explored in the following thesis through conjugation of shark-derived immunoglobulin fragments known as Variable New Antigenic Receptors (VNARs) to the surface of polymeric NPs. This is a novel use of VNARs for which they have many advantageous properties including high physicochemical stability, small size, and low chemical complexity while still maintaining the ability to bind a diverse range of epitopes with high affinity.
The possibility of multiple functional groups being available for targeting ligand conjugation is explored in the first chapter of this work. Polymers bearing vinyl sulfone or sulfonyl fluoride groups were obtained or synthesised, respectively. Both functional groups were incorporated within amphiphilic block copolymers which had not been previously investigated for protein conjugation. Vinyl sulfone or sulfonyl fluoride functionalised polymers were blended with PLGA in separate formulations and C-terminal Cys bearing anti Delta-like ligand 4 (DLL4) VNARs conjugated to the resultant NPs. Their binding capabilities were then examined which, while functional, were not as potent as a more routine maleimide PLGA-PEG NP approach.
Secondly, the potential of anti-DLL4 VNAR conjugated NPs in pancreatic ductal adenocarcinoma (PDAC) relevant models was investigated. Anti-DLL4 VNARs were covalently conjugated to maleimide functionalised PLGA-PEG NPs to produce conjugates with physicochemical characteristics amenable to therapeutic use and with high specific DLL4 binding affinity. Anti-DLL4 VNAR conjugated NPs were found to preferentially associate with DLL4 expressing PDAC cell lines and showed an anti-angiogenic effect in endothelial cells. This latter effect was enhanced by multimerization of VNARs on the NP surface indicating a distinct advantage of the VNAR conjugated NP concept.
Finally, the use of an anti-human serum albumin (HSA) VNAR to control the formation of the NP protein corona in biological media was demonstrated. Formation of a stable HSA corona on NPs then influenced the identity of further corona formation as shown by mass spectrometry. Coronal control also influenced NP cellular interaction, with increased fluorescent NP association and increased camptothecin (CPT) encapsulated NP cytotoxicity observed against PANC-1 cell lines. A pilot in vivo biodistribution study was also performed with fluorescent NPs indicating a different biodistribution profile to naïve control VNAR conjugated NPs and warranting further investigation.
In summary this work within this thesis exemplifies the surface modification of polymeric NPs with VNARs and characterises the therapeutic potential of the VNAR NP conjugates for pancreatic cancer therapy.
|Date of Award||Jul 2021|
|Sponsors||John Martin’s Charity|
|Supervisor||Christopher Scott (Supervisor) & Daniel Longley (Supervisor)|
- Pancreatic cancer
- Variable New Antigen Receptors
- Poly(lactic-co-glycolic) acid
- Delta-like ligand 4
- Nanoparticle corona
- Secreted Protein Acidic and Rich in Cysteine