Fig 1.
Improved uptake of exosomal doxorubicin.
(a) HEK293 cells were incubated with different concentrations of free doxorubicin (Dox) or exosomal doxorubicin (Exo-Dox) in duplicate at 37 °C for 4 h. Uptake was analysed by flow cytometry, average mean fluorescence intensity (MFI) values were calculated and normalised to the MFI value obtained by incubating cells with an equivalent volume of PBS as control (b) HEK293 cells were incubated with free doxorubicin (Dox) or exosomal doxorubicin (Exo-Dox) for 4 h either at 37 °C or on ice in duplicate. Uptake was analysed by flow cytometry as described in a) n = 3; data is presented as mean +/-SD *p<0.05 and ****p<0.0001, ns non significant.
Fig 2.
Rapid internalisation of exosomal doxorubicin.
(a) HEK293 cells were incubated with 0.25 μg/ml Dox, Exo-Dox, liposomal formulations of Dox or PBS as control in duplicate for increasing amounts of time. Doxorubicin uptake was analysed by flow cytometry as described in Fig 1. (b) uptake was performed as described in (a) for 15 min. (c) HEK293 cells were incubated with increasing amounts of Dox, Exo-Dox, liposomal formulations of Dox or PBS for 15 min and uptake was assessed by flow cytometry analysis. (a,b,c) MFI values were normalised to MFI values obtained by incubation in PBS. All experiments were performed n = 3 times, data is presented as mean +/-SD, ****p<0.0001. (d) HEK293 cells were incubated with Dox, Exo-Dox, liposomal formulations of Dox (red) at concentrations indicated for 15 min followed by staining of the nuclei with Hoechst (blue). Uptake was analysed by epifluorescence microscopy; (e) experiment was performed as in (d), images for Dox concentrations that result in comparable recipient cell fluorescence are shown. (d,e) representative images from one (out of three) independent experiments are shown. Scale bar in microscopic images: 10 μm.
Fig 3.
Sustained uptake of exosomal doxorubicin causes a high cumulative concentration.
(a) HEK293 cells were incubated with 0.25 μg/ml Dox, liposomal Dox or Exo-Dox for 4 h at 37 °C or in duplicate. Cellular uptake was analysed as described in Fig 1. (b) HEK293 cells were incubated with increasing amounts of Dox, Exo-Dox, liposomal formulations of Dox or PBS and uptake was determined by flow cytometry analysis. (a,b) MFI values were normalised to MFI values obtained by incubation in PBS. Experiments were performed n = 3 times, data is presented as mean +/-SD, ****p<0.0001. (c) HEK293 cells were treated with Dox, Exo-Dox, liposomal formulations of Dox (red) at concentrations indicated for 4 h followed by Hoechst staining of the nuclei (blue). Uptake was analysed as described in Fig 2. (d) Uptake experiment was performed as in (c), images for Dox concentrations that result in comparable recipient cell fluorescence are shown. Representative images from (one out of three) independent experiments are shown. Scale bar: 10 μm.
Fig 4.
Time-lapse videomicroscopy shows that Exo-Dox causes enhanced mitochondrial swelling and apoptosis.
HEK293 and PASMC were treated with 170 nM Exo-Dox or Dox at the same and a 5-fold higher concentration (850 nM) in triplicate. Mitochondrial morphology was analysed by addition of mitotracker and apoptotic cell death was analysed by addition of caspase 3 sensitive substrate DEVD-nucview488 using time-lapse videomicroscopy for which cells were imaged every 2 h over the course of 30 h. Fig 4a shows representative images taken at the 24 h timepoint of HEK293 (top panel, mitotracker staining) or PASMC cells (bottom panel, caspase 3 substrate staining). Relative fluorescence intensity was determined by divinding fluorescence confluency by phase confluency. Fig 4b shows a timecourse of relative fluorescence intensity for mitochondrial dye mitotracker and caspase 3 activity. One (out of three) representative experiments is shown. Data is displayed as mean +/-SEM; scale bar: 100 μm.
Fig 5.
Exosomal doxorubicin is more potent than free doxorubicin or liposomal formulations of doxorubicin in a wide range of cultured cells.
HEK293, SK-BR-3, BT-20, HUVEC, PASMC and hiPS cardiomyocytes were treated with increasing amounts of Dox, Exo-Dox, Myocet, Doxil or control liposomes in duplicate for 48 h (HEK293, SK-BR-3, BT20) or 72 h (HUVEC, PASMC, iCell cardiomyocytes). Cellular ATP content as measure of cell viability was determined using luminescent cell titre Glo assay. Results were plotted in GraphPad Prism 6 using a 4-parameter logistic equation to give IC50 values. Representative graphs are shown in (a). (b) table summarising mean +/- SEM. IC50 values from n = 3 independent experiments performed in HEK293, SK-BR-3, BT-20, HUVEC and PASMC and n = 2 independent experiments performed in hiPS cardiomyocytes.