Triplicate wells were treated with CCNSs, free etoposide, and ECCNSs in different concentrations of 5, 10, 20, and 40 μg/mL. These SGC-7901 cells were incubated as described above for 24 and 48 h. MTT of 20 μL (5 mg/mL) was added to each well before the cells were incubated for 4 h at 37°C under light-blocking condition. After the removal of the MTT dye solution, cells were treated with 150 μL DMSO. Absorbance was measured at 490 nm using ELX 800 reader, and inhibition against

SGC-7901 cells was calculated by the following equation: Fluorescence activated cell sorter analysis The number of the apoptosis cells was determined with the Annexin V-PI detection kit (KeyGEN Biotech). SGC-7901 cells with 1 × 106 were cultured, suspended in RPMI-1640 with 10% pasteurized FCS, and seeded on a 24-well flat-bottomed plate and incubated for 24 h at 37°C. The free etoposide, ECCNSs, and culture medium were only ARRY-438162 nmr added to each group with

the concentration of 30 μg/mL. Based on the drug encapsulation efficiency, the same quantity of etoposide was applied to all formulations for the apoptosis analysis. The incubation continued for 24 h at 37°C. Then, the cells were harvested and washed with PBS, and then PI and Annexin V were added directly to the cell suspended in the binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). The cells were incubated in the dark for 15 min at 37°C and submitted to FACS analysis on a Beckton-Dickinson (Mountain View, CA, USA) spectrophotometer. Confocal laser scanning microscopy CLSM images of the ECCNSs and etoposide were obtained using confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with an oil immersion selleck kinase inhibitor objective (60×, Zeiss, Oberkochen, Germany). A suspension of the SRT2104 in vitro particles was placed on a glass slide and dried prior to use. Fluorescence images were obtained at an excitation wavelength of 488 nm (fluorescein isothiocyanate Methane monooxygenase (FITC)) and 405 nm (4′,6-diamidino-2-phenylindole (DAPI)). Results and

discussion As shown in Figure 1, CCNSs were obtained by a multistage self-assembled strategy. In this study, a series of intermediates were trapped, in order to confirm the formation process of the CCNSs. It was found that the nanoparticles firstly concentrated and arranged in a line at an early stage. Then, the particles grew rapidly into the broom shape via crystallization of nanoparticles coupled with a simultaneous multiscale assembly. With the reaction going on, the broom-like structure formed into a high-order spherical structure, as shown in Figure 2. The CCNSs were synthesized by a binary solvent method. Firstly, the reaction of citric acid with HCO3 − ions generates CO2 bubbles and H2O. And then, the CO2 bubbles serve as not only the template of engineered nanospheres but also the reactive materials (reaction formulas listed below). Furthermore, citric acid acts as a crystal modifier to control the selectivity of polymorph and crystal morphology.