Cisplatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly(D,L-lactide-co-glycolide) (PLGA, copolymer ratio 75:25, molecular weight = 66,000–92,000), polyvinyl alcohol (PVA, average molecular weight = 30,000–70,000) and curcumin were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, penicillin G and trypsin-EDTA were obtained from Life Technologies (Carlsbad, CA, USA). Caspase-3/-7 and caspase-9 activity assay kits were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The primary antibodies against caspase-3, caspase-9, Apaf-1, cytochrome c, AKT, p-AKT and BAD were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). All other antibodies used in this study and horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit or mouse immunoglobulin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The enhanced chemiluminescence (ECL) detection kit (Immobilon Western Chemiluminescent HRP Substrate) was obtained from Merck Millipore (Billerica, MA, USA).

Human osteosarcoma U2OS cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained at 37°C under a humidified 5% CO₂ atmosphere in 90% McCoy’s 5a medium (Invitrogen Life Technologist, Grand Island, NY, USA) containing 2 mM L-glutamine, 10% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (Life Technologies) (47–50).

Curcumin-loaded PLGA nanoparticles (Cur-NPs) were prepared by using single emulsion solvent evaporation method. Cucurmin (1 mg) and PLGA (10 mg) were dissolved in dichloromethane. The curcumin and PLGA solution (1 ml) was added to 2 ml of 10% (w/v) PVA surfactant solution to form an oil-in-water emulsion by sonication. The emulsion was carried out by setting sonication at 55 W of energy output for 3 min over an ice bath. The formed emulsion was dispersed by dropping into the 0.5% (w/v) PVA solution and stirred for an additional 4 h at room temperature on a magnetic stir plate to allow evaporation of organic solvent. Nanoparticles were collected by centrifugation at 12,000 rpm for 30 min and washed twice with double distilled water to remove PVA and un-encapsulated curcumin. The prepared nanoparticles were collected and lyophilized (42).

The size and the polydispersity of prepared nanoparticles (PLGA-NPs and Cur-NPs) were measured using DLS (Zetasizer Nano ZS, 3000HS, Malvern Instruments Ltd., Worcestershire, UK). To determine the encapsulation efficiency in Cur-NPs before freeze-drying, the amount of non-encapsulated curcumin was measured the absorption value of OD_{450 nm} by ELISA reader. The encapsulation efficiency was calculated by [(Total amount of curcumin- non-encapsulated curcumin)/Total amount of curcumin] × 100% (51).

The morphology of test nanoparticles was examined by TEM (Jeol, Tokyo, Japan). A dilute suspension of nanoparticles (1/10 dilution) was prepared in double distilled water. One drop of this solution was placed on the TEM grid for 10 min, washed twice with double distilled water and allowed to dry overnight. The images were observed and captured at an accelerating voltage of 120 kV under a microscope (42).

NMR spectra were obtained on a Bruker 500 MHz FT-NMR (model: Avance III DPX-500) spectrometer in CDCl3. The following abbreviations are used: s, singlet; d, doublet; m, multiplet. Mass spectra were measured with a Finnigan/Thermo Quest MAT 95XL instrument (52,53).

The cell viability was assessed by the MTT assay. The U2OS cells were cultured in a 96-well plate at the density of 1×10⁴ cells per well and were incubated with 0, 0.25, 0.5, 1 and 2 μg/ml of Cur-NPs for 24 and 48 h. Then, culture medium containing 500 μg/ml MTT was added to each well and incubated at 37°C for 4 h. After incubation, the supernatant was removed. The formed blue formazan crystals in viable U2OS cells were dissolved with isopropanol/0.04 N HCl, followed by measurement of the absorbance of each well at 570 nm with the ELISA reader. All experiments were performed in triplicate. The morphological examination in Cur-NPs-treated U2OS cells was determined under a phase-contrast microscope (50).

To track the internalization of Cur-NPs, U2OS cells (1×10⁶) were seeded on 6-well plates and incubated overnight. Subsequently, cells were treated with 1 μg/ml of Cur-NPs for 24 h. Finally, cells were washed with PBS twice and the internalized curcumin particles were observed under a fluorescence microscope with the filter of 488-nm excitation wavelength and 520-nm emission (27).

The U2OS cells (1×10⁷) were exposed to 1 μg/ml Cur-NPs for 48 h. Cells were harvested, washed by PBS and then lysed in 500 μl lysis buffer at 4°C. The lysed cells were digested overnight with proteinase K (100 μg/ml) at 50°C followed by incubation with 50 μg/ml RNase A at 37°C for 1 h. DNA fragments were extracted twice with phenol/chloroform/isopropanol (24:25:1; v/v/v) and precipitated with 50% isopropanol with glycogen (20 μg/ml) before being re-suspended in 100 μl Tris-EDTA (TE) buffer (Amresco Inc., Solon, OH, USA). Samples were electrophoresed on 1.8% (w/v) agarose gel (Sigma-Aldrich Corp.) in 0.5X TBE buffer (Amresco Inc.) and DNA was stained with 1 μg/ml ethidium bromide (Life Technologies). The gel was observed and photographed under a UV lamp (54,55).

The U2OS cells (1×10⁷) were treated with 0, 0.5, 1 and 2 μg/ml of Cur-NPs for 12 or 48 h. Cells were then harvested, lysed and the total proteins were collected by SDS sample buffer. Approximately 50 μg of proteins from each treatment were resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred to the Immobilon-P Transfer Membrane (Merck Millipore). The transferred membranes were blocked with 5% non-fat dry milk in 20 mM Tris-buffered saline/0.05% Tween-20 for 1 h at room temperature followed by incubation with appropriate primary antibodies at 4°C overnight. At the end of incubation, membranes were washed with Tris-buffered saline/Tween-20 and incubated with secondary antibodies conjugated with HRP. The blots were developed by the chemiluminescence kit and then exposed to X-ray film. Each membrane was stripped and reprobed with anti-β-actin antibody (Sigma-Aldrich Corp.) to ensure equal protein loading during the experiments (47,56,57).

The U2OS cells (1×10⁷) were exposed to 1 μg/ml of Cur-NPs for 0, 12, 24, 36 and 48 h. Subsequently, cells were harvested, and cell lysates were assessed in accordance with the manufacturer’s instructions provided in the caspase-3/-7 and caspase-9 colorimetric assay kits (R&D System Inc.). Cell lysate containing 50 μg proteins were then incubated for 1 h at 37°C with specific caspase-3/-7 substrate (DEVD-pNA) or caspase-9 substrate (LEHD-pNA) and determined by measuring OD₄₀₅ of the released pNA (58–60).

All the statistical results are performed as the mean ± standard error of the mean (SEM) for the indicated numbers of independent experiments. Statistical analyses of data were done using one-way ANOVA followed by Student’s t-test and the levels of P<0.001 was considered significant between the treated and untreated group (61).

In order to improve the application of curcumin, curcumin was encapsulated by PLGA to form curcumin-loaded PLGA nanoparticles (Cur-NPs) by single emulsion method (Fig. 1A). The nanoparticle form of curcumin exhibited good water-solubility and formed a transparent solution while dissolving in water. TEM was used to examine the morphology of the Cur-NPs. As shown, the Cur-NPs showed spherical shape (Fig. 1B). The size distribution of Cur-NPs in aqueous solution was measured by DLS. As shown in Fig. 2A, the size of Cur-NPs is ∼250 nm, similar to the curcumin-empty PLGA-NPs. Both nanoparticles showed small polydispersity index (PDI) (Fig. 2A), indicating their homogeneous size distribution (Fig. 2B). The encapsulation efficiency of curcumin in Cur-NPs prepared by single emulsion was 90.5±3.0% (Fig. 2A).

In order to confirm that curcumin was unaffected after nano-technologization, mass (MS) spectroscopy of curcumin and Cur-NPs were performed and we found the curcumin fragments on MS spectrum of Cur-NPs (Fig. 3). Furthermore, the proton nuclear magnetic resonance (¹H-NMR) spectroscopy was used to obtain the ¹H-NMR spectra of PLGA-NPs [poly(lactic-co-glycolic acid) nanoparticles], curcumin standard and Cur-NPs (Fig. 4). Comparing Fig. 4A and B, all the peaks of curcumin standard (Fig. 4A) could be found on the Cur-NPs spectrum (Fig. 4B) with identical chemical shifts and integration. Besides the peaks of curcumin, there were some other peaks, δ1.58–1.62 (354H, m), 4.66–4.93 (58H, m) and 5.17–5.27 (89H, m), on the Cur-NPs spectrum. The chemical shifts and integration ratio (∼4:0.6:1) of these additional peaks are identical with that of PLGA NPs, δ1.58–1.62 (4H, m), 4.63–4.93 (0.6H, m) and 5.17–5.27 (1H, m). From the careful inspections of spectral data described above, curcumin was constant and unaffected by nano-technologization.

The U2OS cells were treated with Cur-NPs (0, 0.25, 0.5, 1 and 2 μg/ml) for 24 and 48 h. The cells were collected and the cell viability was determined using MTT assay. Our results showed that the concentrations of 0.25, 0.5, 1 and 2 μg/ml Cur-NPs significantly decreased cell viability in U2OS cells concentration- and time-dependently (Fig. 5A). Cellular uptake of Cur-NPs was visualized by green fluorescence of curcumin using fluorescence microscopy (Fig. 5B). Intensified fluorescence was detected in the cytoplasm and nucleus of cells treated with Cur-NPs, suggesting the amount of curcumin internalized into the cells. Our results demonstrated that Cur-NPs display the anti-human osteosarcoma U2OS cells in vitro.

After treatment with 0.5, 1 and 2 μg/ml of Cur-NPs for 48 h, Fig. 6A revealed apoptotic bodies in Cur-NPs-treated U2OS cells and this effect is concentration-dependent. Further results are demonstrated in Fig. 6B, which indicated that Cur-NPs induced DNA fragmentation in Cur-NPs-treated U2OS cells.

To examine whether Cur-NPs induces apoptosis in U2OS cells, cells were treated with 1 μg/ml of Cur-NPs for 0, 12, 24, 36 and 48 h before subjected to caspase-3/-7 and caspase-9 activities. Cur-NPs stimulated caspase-9 (Fig. 7A) and caspase-3/-7 (Fig. 7B) activities in Cur-NPs-treated U2OS cells and this effect is time-dependent. Based on these findings, we provide evidence regarding the intrinsic caspase contributing to Cur-NPs-induced apoptosis in U2OS cells.

We examined the effects of Cur-NPs on mitochondria-dependent and Akt-Bad signaling pathways in U2OS cells. The immunoblotting results showed that the protein level of p-Akt was decreased in Cur-NPs-treated U2OS cells (Fig. 8A). In contrast, the protein levels of cleaved caspase-3, cleaved caspase-9, cytochrome c, Apaf-1 and Bad were increased in Cur-NPs-treated U2OS cells (Fig. 8B). In conclusion, our data expand the current understanding of Cur-NPs treatment in U2OS cells on causing cell death through the mitochondrial-dependent caspase cascade and the Akt-Bad signaling in vitro.