Size Distribution of Curcumin/Liposome.

<p>Representative data on the size of curcumin/liposome are shown here. The vertical axis shows the relative intensity of light scattering and the horizontal axis shows the size of liposomes on log scale.</p

Dose-Response Curves of Curcumin/Liposome in Inhibition of Cell Proliferation.

<p>THP-1 (panel A) or RAW264.7 (panel B) cells were cultured for 3 days in the presence of curcumin/liposome (●), curcumin (▲), and control liposomes (○), respectively. After culturing, they were incubated with WST-8 for 3 h to assess viable cells in the culture. Resultant coloring reaction by WST-8 was measured with a plate-reader at the absorbance of 450 nm. In the vertical axis, the absorbance of only the medium was defined as 0%. Symbols in the Figure represent the percentage of absorbance with curucmin or liposomes to that without the test samples. Data represent mean values ± range (bars) in duplicate assays.</p

Effect of In Vivo Administration of Curcumin/Liposome on IL-6 Production from Mouse Peritoneal Cells.

<p>Balb/c mice were injected with thioglycollate 3 days before harvesting peritoneal cells. Curcumin/liposome (filled bars) or control liposomes (gray bars) were injected into the mice 4 or 24 h before harvesting the cells. The harvested cells were cultured for 24 h in the absence (panel A) or presence (panel B) of LPS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137207#pone.0137207.g004" target="_blank">Fig 4</a>. Blank columns represent IL-6 production in the culture of the peritoneal cells from mice without receiving the injection of liposomes. IL-6 in the culture supernatants were quantified in triplicate assays. Data represent mean values ± standard deviation (bars) in triplicate assays. **indicate that P<0.01 compared to mice injected with control liposomes.</p

Effect of Curcumin/Liposome on IL-6 Production from Mouse Peritoneal Cells in In Vitro Culture.

<p>Peritoneal cells harvested from Balb/c mice injected with thioglycollate 3 days before. Peritoneal cells were cultured for 24 h in the absence (panel A) or presence (panel B) of LPS at 0.5 μg/ml. Blank columns represent cultures absent of both curcumin/liposome and control liposome. Curcumin/liposome (filled bars) was added to the culture so that curcumin concentrations became as indicated in the Figures. Control liposomes (gray bars) were added to culture in the same manner as curcumin/liposome, and its lipid concentration was consistent with that of curcumin/liposome. Data represent mean values ± standard deviation (bars) in triplicate assays. The statistical analysis was carried out by the standard Student’s t-test. * and **indicate that P<0.05 and P<0.01 compared to control liposomes, respectively.</p

Effect of Curcumin/Liposome Administration on Body Weight Changes in Mice.

<p>Balb/c mice were injected i.p. (panel A) or i.v. (panel B) with curcumin/liposome suspension (200 μl). ○: untreated mice; ●: mice injected with curcumin/liposome (2.9 mg/ml). Data are shown as mean values ± standard deviation (bars) in five mice. The statistical analysis was carried out by the standard Student’s t-test. * and **indicate that P<0.05 and P<0.01 compared to untreated mice, respectively.</p

Nanoparticles Containing Curcumin Useful for Suppressing Macrophages <i>In Vivo</i> in Mice

<div><p>To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice.</p></div

Representative Compounds Showing Inhibitory Activities in Cell Proliferation Assays.

<p>^aTest samples were dissolved in DMSO and then added to cell culture. Final concentration of DMSO was adjusted less than 1% in the culture. Under this condition, DMSO did not show inhibitory activities.</p><p>^bDose-response curves were obtained by two-fold serial dilution of test samples starting from 400 mM at most. In each experiment, each assay was done in duplicate. Data represents means ± range (n = 2) or standard deviation (n > 2) in repeated experiments.</p><p>^cn represents the number of experiments done to obtain these data.</p><p>Representative Compounds Showing Inhibitory Activities in Cell Proliferation Assays.</p

ASSQ percentiles.

<p>ASSQ percentiles.</p

Inpact of ASSQ cutoff score on sensitivity and specificity.

<p>Inpact of ASSQ cutoff score on sensitivity and specificity.</p

Internal consistency of the ASSQ.

<p>Internal consistency of the ASSQ.</p