The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery.
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.
Richardson
Simon C W
SC
Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Av, Cardiff, Wales CF10 3XF, UK. S.C.W.Richardson@greenwich.ac.uk
Wallom
Kerri-Lee
KL
Ferguson
Elaine L
EL
Deacon
Samuel P E
SP
Davies
Matthew W
MW
Powell
Alison J
AJ
Piper
Robert C
RC
Duncan
Ruth
R
D000178:Acrylamides / Q000493:pharmacokinetics*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002522:Chlorocebus aethiops; D003912:Dextrins / Q000493:pharmacokinetics*; D004337:Drug Carriers / Q000493:pharmacokinetics*; D011992:Endosomes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D049329:Nanostructures; D004364:Pharmaceutical Preparations / Q000008:administration & dosage*; D011092:Polyethylene Glycols / Q000493:pharmacokinetics*; D051381:Rats; D012995:Solubility
D000178:Acrylamides; D003912:Dextrins; D004337:Drug Carriers; D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; C032976:N-(2-hydroxypropyl)methacrylamide