The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery
Richardson, Simon C.W., Wallom, Kerri-Lee, Ferguson, Elaine L., Deacon, Samuel P.E., Davies, Matthew W., Powell, Alison J., Piper, Robert C. and Duncan, Ruth (2008) The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery. Journal of Controlled Release, 127 (1). pp. 1-11. ISSN 0168-3659 (doi:10.1016/j.jconrel.2007.12.015)Full text not available from this repository.
Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.
|Additional Information:|| Available online 4 January 2008. Published in Journal of Controlled Release, Volume 127, Issue 1, 7 April 2008.  Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2007.12.015.|
|Uncontrolled Keywords:||endocytosis, polymer conjugates, nanomedicine, intracellular trafficking|
|Subjects:||R Medicine > RS Pharmacy and materia medica|
|Faculty / Department / Research Groups:||Faculty of Engineering & Science > Department of Pharmaceutical, Chemical & Environmental Sciences|
|Last Modified:||17 Oct 2016 09:10|
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