Anane-Adjei, Akosua Bema
(2020)
Investigation of polymer drug conjugates for pancreatic cancer.
PhD thesis, University of Nottingham.
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal forms of cancer with a 10-year survival rate of <1 %. Although considerable advancement has been made in understanding the biology of the disease, most patients presenting with locally advanced or metastatic disease have an extremely poor prognosis. Gemcitabine (GEM), a nucleoside analogue of cytidine is the first-line treatment for patients diagnosed with locally advanced or metastatic disease. However, approx. 90 % of the intravenously administered dose of GEM undergoes rapid deamination in the bloodstream by cytidine deaminase (CDA), affecting the drug’s therapeutic efficiency. Due to this, large doses are needed to achieve significant therapeutic efficacy leading to increased adverse effects.
In the work presented here, we report on the synthesis of hyperbranched N-(2-hydroxypropyl)methacrylamide) (HPMA) polymer-drug conjugates for improved biodistribution and pharmacokinetic profiles of the administered drug(s). The polymers were evaluated in two-dimensional (2D) and three-dimensional (3D) cell culture models of MIA PaCa-2 pancreatic cancer cell line.
In chapter 3, we showed the development and characterisation of the 3D pancreatic cancer models used in this work for assessing the efficacy of the drug delivery system. The MIA PaCa-2 cells were mono and cocultured with mesenchymal stem cells (MSCs) and characterised for growth, the viability of the cells in the spheroid and MSCs retention in order to select the optimum conditions for all subsequent spheroid experiments. From this experiment, we determined that 1000 cell/well and 1:1 (1000 cells each) ratio were the ideal seeding densities for the mono and coculture models of the pancreatic cancer line respectively.
In chapter 4, we synthesised three hyperbranched HPMA polymers with different hydrodynamic diameters (7 nm, 20 nm and 40 nm) to determine the ideal size for our system. All three polymers were found to be biocompatible with macrophage and the MIA PaCa-2 pancreatic cancer cell line. They were also found to exhibit ‘stealth-like’ properties with low uptake into RAW 264.7 macrophage cells. The biodistribution studies show differences between the fluorescent and PET-CT assays. The small-sized polymer (7 nm) was found to accumulate more in the kidneys than the 20 nm and 40 nm diameter polymers in the fluorescent studies, which is thought to be due to the small diameter of the glomerular capillary. However, in the PET-CT assay, all three polymers had enhanced liver and spleen uptake with the smallest polymer having the highest accumulation in these tissues. This is thought to be due to potential conformational changes to the polymers as a result of the conjugation of the deferoxamine (DFO) chelator. The 20 nm hydrodynamic diameter was chosen for the development of our polymer-drug conjugate systems as it exhibited the most desirable characteristics.
Chapters 5 and 6 focus on the development of our polymer-drug conjugates. Three polymer conjugates were synthesised (HPMA-GEM, HPMA-DOX and HPMA-DOX-GEM) for mono and combination therapy and evaluated in 2D and 3D cell culture models. The results show that the IC50 of the free drugs (gemcitabine and doxorubicin) was significantly lower than the IC50 of their respective polymer conjugates in the 2D cell culture model. Observed toxicities in the cells showed the polymer conjugates could deliver the chemotherapeutic agents effectively. In 3D cell culture, we saw less pronounced differences in efficacy between the free drugs and conjugated drugs. This may be due to improved polymer penetration through the spheroids over the small molecule free drugs. The efficacy of the polymer-drug conjugates especially in the 3D cell culture makes our hyperbranched HPMA polymer a promising delivery system for pancreatic cancer. In combination therapy, the two drugs were found to have a synergistic effect on the pancreatic cancer cells in the 2D cell culture model. However, in the 3D cell culture assay, the drugs were shown to display antagonistic effect potentially due to the reduced effectiveness of doxorubicin in the combination. This observed difference could be attributed to the inability of the 2D cell culture model to accurately predict the in vivo behaviour as a result of the lack of complexity of the model; demonstrating the need to adopt 3D cell culture models for the screening of cancer therapeutics.
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