Fluorescence microscopy studies were conducted to test the intensity of uptake by MCF-7 cells of the encapsulated Nile red. The uptake was significantly faster for Nile Red-CS nanocapsules than for the other two types. After only 1 h, CS particles showed the highest uptake, followed by Epi and then PhS particles (Figure 1). Uptake increased with longer incubation time, preserving these differences among particles until 5 h, when the cells showed the same amount of fluorescence, and no differences among the three types of nanoparticles could be detected. At 3 h of incubation, the Nile red was localized in the cytoplasm of cells (Figure 1), indicating that these nanosystems are effective vehicles to transport the molecules within MCF-7 cells by endocytosis, and can avoid efflux by cell membrane MDR transport proteins such as p-glycoprotein (P-gp) [25,27,28]. The variation in internalization among the different nanoparticles may be attributable to a combination of surface charge and hydrophilicity, which play a major role in affinity in the endocytosis pathway [29]. The CS nanocapsules showed the highest fluorescence intensity, related to their positive superficial charge, which increases the affinity between nanoparticle and negatively-charged cell membrane [30]. This finding is consistent with the enhanced mucoadhesive properties reported for chitosan nanosystems [31,32]. The lower fluorescence response observed with the EPI and PhS systems appears attributable to the size and negative surface charge of these nanocapsules, as well as to the hydrophilic nature of the poloxamer molecules, which produce systems that are more biocompatible but have a lower cell uptake [33,34]. The lower fluorescence intensity of PhS versus EPI nanocapsules may be attributable to the lesser hydrophilicity of the former. Nevertheless, given that these differences disappear after 5 h of treatment, all three systems achieve similar uptake efficacy (data not shown).

Docetaxel-loaded nanoparticles were prepared by using a solvent-displacement technique. For encapsulation, docetaxel was dissolved in olive oil from the organic phase of the emulsion before it was mixed with the aqueous phase. High-performance liquid chromatography (HPLC) study of the encapsulation efficiency of docetaxel showed that the organic phase of CS, Epi, and PhS nanoparticles contained 86%, 80%, and 80% of the drug, respectively. In cytotoxicity assays, MCF-7 cells were treated with free docetaxel or increasing concentrations of the different docetaxel-loaded nanocapsules for 6 days, followed by calculation of the inhibitory concentration 50 (IC₅₀) in each case. No significant differences in growth patterns were found between parental MCF-7 cells and MCF-7 cells treated with empty CS, Epi, or PhS nanoparticles (data not shown). With equivalent drug loading in the culture medium, a markedly lower proliferation rate was observed in the MCF-7 cells treated with docetaxel-loaded nanoparticles than in the parental MCF-7 cells treated with free docetaxel (Figure 2). A 20 nM dose of free docetaxel had no cytotoxic effect on cells, whereas 20 nM of docetaxel-loaded CS, Epi, or PhS nanoparticles induced a significant decrease of 94.65%, 89.27%, or 51.01%, respectively, in their survival rate (P < 0.001) (Figure 2a). IC₅₀ values were 45 nM for free docetaxel, 9 nM for docetaxel-loaded CS and Epi nanoparticles, and 20 nM for docetaxel-loaded PhS nanoparticles (Figure 2b).

Recent studies reported similar effects with high-dose docetaxel in oily core nanocapsules. Youm et al. [35] observed that the bioactivity against SUM 225 cells was higher for free versus encapsulated docetaxel at lower concentrations (2.5 μM), but was significantly higher for the encapsulated form at the highest concentration (5 μM). Likewise, Li et al. [36] found that cell death was greater in drug-resistant MCF-7/ADR cells when doxorubicin was delivered in LA-TPGS nanoparticles at high drug concentrations. Importantly, the Epi, CS, and PhS nanocapsules described in the present study induced high cell death levels even at low concentrations, exerting a five-fold (Epi and CS) and 2.26-fold (PhS) more potent cytotoxic effect versus free docetaxel. These data indicate that the sustained release of the drug and its enhanced drug internalization by MCF-7 cells when in this nanocapsule formulation may increase the biological response to docetaxel and allow a decrease in the dose, thereby reducing its side effects.

Apoptosis is considered the main cell death mechanism in response to taxanes. Docetaxel targets tubulin, stabilizing microtubules and thereby inducing cell-cycle arrest and apoptosis [37]. We therefore studied these well-documented effects on cell cycle distribution and apoptosis in the tumor cells. When undergoing apoptosis, cells externalize phosphatidylserine, a lipid found on the inner surface of the cell membrane that can be labeled with fluorochrome-conjugated annexin V. It is therefore possible to distinguish between intact cells (stained negative for both annexin V-FITC and propidium iodide [PI]), early apoptosis (stained positive for annexin V-FITC and negative for PI), late apoptosis or cell death (stained positive for both annexin V-FITC and PI), and necrosis (stained positive for PI).

Our flow cytometry study with annexin V assay showed that 94.30% of control MCF-7 culture cells were viable, 1.05% were early apoptotic, 0.35% were in late or final stages of apoptosis, and 4.30% were necrotic (P < 0.001) (Figure 3). No significant differences versus control cells were found in MCF-7 cells treated for 24 h with empty nanoparticles (data not shown). No apoptosis was detected in cells treated with 45.2 nM free docetaxel for 24 h. Significant apoptosis levels were observed in cells treated with docetaxel-loaded nanoparticles, and the highest apoptosis level (16.19%) was in the cells treated with 9.1 nM of Epi docetaxel-loaded nanoparticles (Figure 3a–e). The confocal microscopy study confirmed these results (Figure 3f–j). In fact, it is known to be difficult to demonstrate programmed cell death by known apoptosis-inducing agents in the MCF-7 human breast cancer cell line, and only a few cytotoxic agents have been found to act preferentially via an apoptotic mechanism in human breast cancer cells [38,39].

It has also been reported that docetaxel acts at molecular level by impairing mitosis and inducing cell-cycle arrest [40]. We performed flow cytometry studies to determine differences in cell cycle distribution among the treatments. Figure 4 depicts the results of treating MCF-7 cells for 24 h with free or nanocapsule-loaded docetaxel at IC₅₀ values. All treatments induced accumulation in G₂/M and S phases, with a significant decrease in G₀/G₁ phase versus control cells. The accumulation was significantly greater in cells treated with docetaxel-loaded nanoparticles than in cells treated with the free drug (Figure 4). The rapid decrease in cell cycle progression, evidenced by the increased percentage of cells in S and G₂/M phase, is in agreement with previous reports that docetaxel treatment induces a G₂ cell cycle buildup in several cell lines, including MCF-7 [41–43].

The human breast cancer MCF-7 cell line was grown at 37 °C in an atmosphere containing 5% CO₂ with Dulbecco’s modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco) 2% l-glutamine, 2.7% sodium bicarbonate, 1% Hepes buffer, and 1% penicillin/streptomycin solution (GPS, Sigma).

Lipid nanosystems were prepared and characterized as described in [16]. Three systems were formulated: EPI nanocapsules, with surface shell composed of Epikuron and Pluronic^® F68; CS nanocapsules, with shell of Epikuron and chitosan oligomers; and PhS nanocapsules, with shell of phosphatidyl-l-serine and Pluronic^® F68. For nanosystem characterization, the average size was determined by combining a high-performance-particle-sizer technology with a non-invasive-backscattering method, in which the measurement is taken at an angle of 173°. A Zetasizer-Nano analyzer (Malvern Instruments, Worcestershire, UK) was used to measure the electrophoretic mobility (μ_e). Zeta potential was calculated from this experimental magnitude in physiological medium conditions.

Fluorescent lipid nanocapsules were formulated by dissolving Nile Red in the olive oil phase at a concentration of 0.025% (w/w). Encapsulation of this fluorescent molecule was confirmed by using a SPEX Fluoromax-2 spectrofluorometer. Emission fluorescence spectra were determined from 550 to 700 nm at an excitation wavelength of 485 nm, with a scanning speed of 100 nm/min and wavelength accuracy of ±1 nm.

Docetaxel-loaded lipid nanocapsules were formulated by dissolving docetaxel in the olive oil phase at a concentration of 0.1% (m/w). The encapsulation efficiency was calculated by HPLC at the Scientific Instrumentation Center of the University of Granada, using a SHIMADZU LC-20AC chromatograph with SPD-M20A diode array detector and C8 Nova-Pak Cartridge column (4 microns, 4.6 × 150 mm); detection was performed at a wavelength of 230 nm.

MCF-7 cells (5 × 10³) were seeded into 6-well plates under the culture conditions detailed above. After 24 h, cells were fed with fresh medium and treated with Nile Red nanocapsules. Cells were incubated with Nile Red labeled particles for 1 h and 5 h and then washed twice with acidic phosphate saline buffer (PBS) to remove free nanocapsules. Fresh PBS was added and living cells were analyzed by fluorescent microscopy (Nikon Eclipse Ti-S). Cells treated with empty nanoparticles were used as controls.

MCF-7 cells (1 × 10³) were plated into 6-well plates under the culture conditions detailed above. The cells were fed with fresh medium and increased concentrations of free and encapsulated drug every alternate day up to the end of the experiment. After 6 days of treatment, cells were counted using sulforodamine-B (SRB) colorimetric assay as previously described [44] using a Titertek Multiscan apparatus (Flow, Irvine, CA, USA) at 492 nm.

We evaluated the linearity of the SRB assay with cell number using stock MCF-7 cells before each cell growth experiment. IC₅₀ values were calculated from semi-logarithmic dose-response curves by linear interpolation. All experiments were plated in triplicate wells and were carried out at least twice.

The Annexin V-FITC Apoptosis Detection kit I (Pharmingen, San Diego, CA, USA) was used to detect apoptosis by flow cytometry. MCF-7 cells (1 × 10⁶ cells) were plated onto 75-cm² flasks and cultured overnight, followed by incubation with IC₅₀ values of free or encapsulated docetaxel for 24 h. Cells were harvested by PBS-EDTA, washed twice in cold PBS (1.4 M NaCl, 27 mM KCl, 100 mM KH₂PO₄/K₂HPO₄, pH 7.2), and pelleted by centrifugation at 500 g for 10 min. They were then resuspended at 10⁶ cells/100 μL in a binding buffer (Hepes buffer, 10 mM, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂), stained with annexin V incubation reagent (1 μL annexin V-FITC (25 μg/mL), 10 μL binding buffer, 10 μL PI (50 μg/mL), and 79 μL H₂O) and incubated in the dark for 15 min at room temperature. Then, 500 μL binding buffer was added and the cells (10,000 cells per sample) were immediately analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). All experiments were performed in triplicate and yielded similar results.

Cells at 70% of confluence were treated with the half maximal inhibitory concentration (IC₅₀) of free or encapsulated docetaxel. Cells in monolayer culture were harvested, washed twice with sample buffer (100 mg glucose; 100 mL PBS without Ca²⁺ or Mg²⁺), and fixed in 70% (v/v) cold ethanol for up to 1 week. Cells were pelleted, washed once with sample buffer, and resuspended in PI solution (50 μg/mL PI, 0.5 mg/mL RNase in sample buffer, pH 7.4) for 30 min in the dark. Fluorescence activated cell sorting (FACS) analysis was performed after 24 h of treatment. All experiments were performed in triplicate and yielded similar results.