Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging.
As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep) density gradient optimised to 5-20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-beta-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation of polymer therapeutics in the endocytic pathway.
Manunta
Maria
M
Welsh School of Pharmacy, Centre for Polymer Therapeutics, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3XF, UK.
Izzo
Lorella
L
Duncan
Ruth
R
Jones
Arwyn Tomos
AT
D000118:Acetylglucosaminidase / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004396:Coloring Agents; D003911:Dextrans / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D020143:Gangliosidoses, GM2 / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011091:Polyesters / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism* / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism*; D014343:Trypan Blue; D033921:Vesicular Transport Proteins / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
D000118:Acetylglucosaminidase; D004396:Coloring Agents; D003911:Dextrans; D007770:L-Lactate Dehydrogenase; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D011091:Polyesters; D011092:Polyethylene Glycols; D011108:Polymers; D014343:Trypan Blue; D033921:Vesicular Transport Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C093846:early endosome antigen 1