Fig 1.
Representative scanning electron micrograph of R-PE-loaded calcium phosphate nanoparticles.
Fig 2.
Loading in nanoparticles enables R-PE to enter cells.
HeLa cells were incubated with R-PE-loaded nanoparticles (CaP/PEI/R-PE) or dissolved R-PE for 3 h (top row) or 6 h (center row). Cells were fixed and stained with phalloidin (green; actin filaments) and DAPI (blue; nucleus). Bottom row: Magnification of the upper images (white boxes; top row): 3 h incubation R-PE/Phalloidin/DAPI for CaP/PEI/R-PE nanoparticles (left) and 3 h incubation R-PE/Phalloidin/DAPI for R-PE alone (right). All scale bars are 20 μm.
Fig 3.
CaP/PEI/R-PE nanoparticles enter HeLa cells and co-localize with early endosomes and lysosomes.
(A) Confocal laser scanning microscopy on HeLa cells after 6 h of incubation with either CaP/PEI/R-PE nanoparticles or untreated, followed by washing with PBS, fixation and staining with EEA1 (green) or Lamp1 (green) and Hoechst33342 (blue). The overall pattern of early endosomes or lysosomes is not affected by incubation with CaP/PEI/R-PE nanoparticles. Scale bar, 20 μm. (B) Higher resolution images to analyse the co-localization of R-PE with EEA1 or Lamp1. HeLa cells were treated and processed as in (A). An enlargement of the boxed area is shown. Diagram on the right: Quantification of R-PE vesicles that are positive for EAA1 or Lamp1. Data represent mean ± SD from three independent experiments (student’s t-test). **, p<0.01. Scale bar 10 μm.
Fig 4.
CaP/PEI/R-PE nanoparticles are degraded in lysosomes after uptake into HeLa cells.
(A) R-PE signal persists after inhibition of lysosomal degradation. HeLa cells were incubated with CaP/PEI/R-PE nanoparticles for 6 h, washed and chased for the indicated time either in the presence of Bafilomycin A1 or without any additive (control). The fluorescence signal of R-PE is shown alone or merged with phase contrast to visualize the cells. Scale bar 50 μm. (B) Quantification of R-PE positive cells from three independent experiments. Data represent mean ± SD (student’s t-test). *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 5.
CaP/PEI/R-PE nanoparticles are not degraded in lysosomes after uptake into HEK293T cells.
There is no degradation of R-PE after cellular uptake as indicated by the persistent fluorescence (control). HEK293T cells were incubated with CaP/PEI/R-PE nanoparticles for 6 h, washed and chased for the indicated time either in the presence of Bafilomycin A1 or without any additive (control). The fluorescence signal of R-PE is shown alone or merged with phase contrast to visualize the cells. Scale bar 50 μm.
Fig 6.
CaP/PEI/R-PE nanoparticles are not degraded in lysosomes after uptake into MG-63 cells.
There is no degradation of R-PE as indicated by the persistent fluorescence intensity (control). MG-63 cells were incubated with CaP/PEI/R-PE nanoparticles for 6 h, washed and chased for the indicated time either in the presence of Bafilomycin A1 or without any additive (control). The fluorescence signal of R-PE is shown alone or merged with phase contrast to visualize the cells. Scale bar 50 μm.
Fig 7.
CaP/PEI/R-PE nanoparticles are degraded in lysosomes after uptake into MC3T3 cells.
After cellular uptake, R-PE is rapidly degraded as indicated by the vanishing fluorescence intensity (control). MC3T3 cells were incubated with CaP/PEI/R-PE nanoparticles for 6 h, washed and chased for the indicated time either in the presence of Bafilomycin A1 or without any additive (control). The fluorescence signal of R-PE is shown alone or merged with phase contrast to visualize the cells. Scale bar 50 μm.