Endocytosis and trafficking of polymer therapeutics in melanoma cells

Seib, Friedrich Philipp 2005. Endocytosis and trafficking of polymer therapeutics in melanoma cells. PhD Thesis, Cardiff University.

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Abstract

The first aim of this study was to establish a subcellular fractionation method to quantify intracellular trafficking of a polymeric anticancer conjugate (HPMA copolymer doxorubicin- PK1). B16F10 murine melanoma cells were chosen for this study as they have been widely used in vitro and in vivo to study the antitumour activity of PK1 and other polymer-drug conjugates. It was first necessary to define a cell breakage assay. The cell cracker achieved a 90% cell breakage efficiency which ensured that subsequent subcellular fractionation experiments were representative for the cell population. Next, markers for nuclei (DNA), mitochondria (succinate dehydrogenase), lysosomes (N-acetyl-beta-glucosaminidase), plasma membrane (alkaline phosphatase) and cytosol (lactate dehydrogenase) were validated and used in due course for the quantitative characterisation of subcellular fractions. Finally, a differential centrifugation method was devised to separate and enrich nuclei (2.2 fold), mitochondria (4.1 fold), endosomes/lysosomes (3.7 fold) and cytosol (2.5 fold). The intracellular trafficking of PK1 was studied by differential centrifugation and time-dependent accumulation in lysosomes was confirmed, in contrast to free doxorubicin which accumulated in the nucleus. In the second part of this study the endocytic properties of polycations used as non-viral vectors for gene delivery were examined. Linear and branched polyethylenimine (PEI, Mw = 25,000 g/mol) and polyamidoamine dendrimers (PAMAM) of generations (G) 2 -- 4 were studied by flow cytometry using Oregon Green functionalised polymers. At non-toxic concentrations maximum internalisation was observed for PAMAM G4, followed by linear and branched PEI achieving 27 -- 36% of the maximum respectively. Lowest uptake was observed for PAMAM G2 and G3. For all dendrimers extracellular binding was between 16 -- 26% of total cell-association (G2 > G3 > G4). In preliminary studies PAMAM G4 and branched PEI were predominately internalised via cholesterol-dependent pathways, whereas internalisation of linear PEIappeared to be independent of clathrin and cholesterol.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Pharmacy
Subjects:	R Medicine > RS Pharmacy and materia medica
Date of First Compliant Deposit:	30 March 2016
Last Modified:	24 Jul 2023 13:50
URI:	https://orca.cardiff.ac.uk/id/eprint/55512

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