br Wang et al also observed microtubule
Wang et al.  also observed microtubule condensation in the unstructured nuclei of the cells with spindle multipolarization in prostate cancer cells exposed to similar doses of PTX (50 nM). These results not only indicate that NP encapsulation of the drug has no eﬀect
on its antimitotic mechanism of action but also that the use of PLGA-PTX NPs may increase the drug’s antimitotic eﬀect. Cells undergoing mitosis could still be observed after exposure to blank PLGA NPs, which did not produce diﬀerences with respect to the control cells.
Additionally, we analysed modulation of the cell death mechanism induced by encapsulated PTX (PLGA-PTX NPs) using apoptosis studies. A higher percentage of cells treated with PLGA-PTX NPs presented ei-ther early or late apoptosis (between 20% and 60% depending on the cell line) compared to those treated with free PTX (Fig. S5). In fact, a similar proportion of LL2, L132 and NCI-H520 cells treated with PLGA-PTX NPs developed early and late apoptosis. Specifically, PLGA-PTX-treated LL2 and L132 cells experienced an increase in early apoptosis of 49% and 12% against free PTX, respectively, while late apoptosis in-creased by 20% in both lines. The NCI-H520 cell line showed almost no diﬀerences in the percentage of early and late apoptosis between both treatments. However, in A549 and NCI-H460 cells, only early apoptosis was observed, being 24% and 32% higher than in cells treated with free PTX, respectively. A similar eﬀect was described using PLGA-PTX NPs coated with chitosan in A549 and H1299 lung cancer cells (25.84% and 12.24% apoptosis at 48 h, respectively). These percentages increased when a peptide coupled to the NPs which targeted the integrin αvβ3
J. Jiménez-López et al.
Fig. 3. Modulation of Minocycline HCl ana-lysis by PTX-loaded PLGA NPs (A). A549 (a), LL2 (b), NCI-H520 (c), NCI-H460 (d) and L132 (e) cell lines were treated with free PTX, PTX-loaded PLGA NPs and blank PLGA NPs at a dose of IC50. FACScan analysis results were expressed as the percentage of labelled cells in each cell cycle phase. Data represent the mean value ± SD of triplicate cultures. *Data with sig-nificant diﬀerences between the treat-ment with PLGA-PTX and free PTX (p < 005). Anti-alpha-tubulin im-munofluorescence analysis (B). Untreated A549, LL2 and L132 cell lines (control) (a), cells treated with PTX (b), blank PLGA NPs (b) and PLGA-PTX (d) were stained with an anti-α-tubulin antibody and Hoechst 33258 (see Material and methods). Characteristic spindle multipolarization of the antimitotic eﬀect of PTX can be observed (arrows). Magnification, 20 × . (C) Detail of A-549 cells (b and d images) to appreciate spindle multi-polarization.
3.6. Intracellular uptake of PLGA nanoparticles and paclitaxel
We used a nile red (NR) conjugated PLGA NP (see Method) to de-termine the improvement of the intracellular uptake of PTX. A549 cells were incubated with free NR and PLGA-NR NPs for diﬀerent periods. Fluorescence microscopy analysis revealed a higher uptake of NR in
cells treated with PLGA-NR NPs compared to those treated with free NR. In fact, a greater accumulation of the fluorescent dye was observed in the cells cytoplasm, whereas accumulation was low in cell nuclei for all exposure times (Fig. 4).
against lung cancer cells, since PTX encapsulation helps the drug enter the cell after very short exposure times (0.5 h). Some studies suggest that PLGA NPs could be internalised by clathrin-mediated endocytosis or pinocytosis, thus escaping the endolysosomes and penetrating into the cytosol after only 10 min of incubation [19,43]. To complete the internalisation studies we determined intracellular PTX concentration after treatment with free PTX and PLGA-PTX NPs. As shown in Fig. 5, intracellular PTX concentration increased only 30 min after drug exposure and was significantly higher in A549 cells with PLGA-PTX NPs (30-fold) than with free PTX. Similar results were ob-tained in LL2 and L132 cells (10.3 and 12.4-fold, respectively).