br All cell lines were seeded
All cell lines were seeded into 6-well plates at a density of 5 × 104 cells/well in A549 and NCI-H460 and 4 × 104, 2.5 × 105 and 6 × 104 cells/well of LL2, NCI-H520 and L132, respectively. 24 h post-seeding, the medium was replaced by serum-free culture medium serum free to synchronize the cell cycles of the cells. After 24 h, serum-free culture medium was replaced by complete culture medium containing the treatments: free PTX, PLGA-PTX and blank PLGA NPs (at the dose corresponding to the IC50 of PTX at 96 h) for 48 h. Cell cycle analysis was performed by flow cytometry using PI/RNase Solution Kit of Inmunostep (Salamanca, Spain). Cells were harvested with PBS-EDTA solution:Trypsin (1:1 v/v) and fixed by adding 70% ethanol in agita-tion. Then, these were incubated for 45 min at 4 °C. Washing with PBS was performed and an extraction solution of the kit (0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8) (1:1 v/v) was added. After 10 min at 37 °C, the kit labelling solution containing RNase, propidium iodide (PI) and PBS was added and the samples were incubated for another 30 min at RT in the dark. Finally, the samples were analysed with a FACScan flow cytometer using FlowJo software.
2.12. Immunofluorescence assays
Cell lines were seeded with DMEM medium into 24-well plates at a
2.13. Apoptosis analysis
After PTX, PLGA-PTX and blank PLGA NPs treatments of the cell lines (see “cell cycle analysis”) an Annexin V-PE Apoptosis Detection Kit of Inmunostep was used. Cells were detached with PBS-EDTA solution:Trypsin (1:1 v/v). Washing with PBS was performed and a solution of Annexin-binding buﬀer, Annexin V-PE and 7 AAD was added. After 15 min at RT in the dark, the Annexin-binding buﬀer was added and the samples were analysed with a FACScan flow cytometer using FlowJo software.
2.14. Intracellular uptake of nile red
The incorporation of NR (free and PLGA-NR NPs) into the Puromycin was quantified in A549 cell line by FACSan flow cytometer. Cell were plated in a 6-well plate at a density of 5 × 104 cells per well. After 12 h, the culture medium was replaced with fresh medium. Then, PLGA-NR NPs and free NR (0.5 μM) were added to the culture medium. Cells were incubated at diﬀerent times (0.5, 1, 2 and 4 h), washed with PBS, trypsinized and centrifuged (1720 rpm) for 5 min. Two washes with PBS were performed before the fluorescence analysis of NR by detection of Phycoerythrin (PE) with FACSan cytometer. In addition, fluorescence microscopy (Leica Leica DM IL LED Microsystems, Wetzlar, German) was used to visualize the intracellular uptake of NR in A549 cell line.
2.15. Intracellular pharmacokinetics of paclitaxel
The concentration of PTX internalised in the cells was measured following a modification of the protocol described by Li et al. . A549, L132 and LL2 were seeded in 6-well plates at a density of 4 × 105 cells/well in DMEM medium. After adhesion to the plate, the cells were treated with free PTX and PLGA-PTX NPs (500 nM). The medium was discarded at 0.5, 1, 2 and 4 h and cells were washed twice with PBS. Cells were resuspended in lysis buﬀer (800 μl) for 5 min. Collected cells were sonicated for 5 min and then, 500 μl of Methyl tert-butyl ether (MTBE), that facilitates the extraction of PTX, and 15 μl of internal standard (docetaxel) were added to each of the samples. After mixing vigorously for 5 min, the samples were centrifuged at 13,200 rpm for 5 min. The organic phase where PTX is found was collected and the samples were evaporated, as described by Fernández-Peralbo et al. , for analysis by ultraperformance liquid chromatography (Wa-tersT, Acquity H Class model) coupled to a triple quadrupole mass spectrometer (Waterrs, XEVO TQ-S model) (UPLC-MS/MS).