Preparation, cellular uptake and angiogenic suppression of shikonin-containing liposomes in vitro and in vivo

Shikonin has anticancer activity, but it has not yet been applied into clinical use. In the present study, shikonin was prepared using liposomes. We aimed to examine several aspects of sh-L (shikonin-containing liposomes): preparation, angiogenic suppression and cellular uptake through self-fluorescence. Sh-L were prepared using soybean phospholipid and cholesterol to form the membrane and shikonin was encapsulated into the phospholipid membrane. Three liposomes were prepared with shikonin. They had red fluorescence and were analysed using a flow cytometer. Angiogenic suppression of sh-L was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], Transwell tests, chick CAM (chorioallantoic membrane) and Matrigel™ plug assay. MTT assay showed the median IC(50) (inhibitory concentrations) as follows: shikonin, sh-L(1) and sh-L(2) were 4.99±0.23, 5.81±0.57 and 7.17±0.69 μM, respectively. The inhibition rates of migration were 53.58±7.05, 46.56±4.36 and 41.19±3.59% for 3.15 μM shikonin, sh-L(1) and sh-L(2), respectively. The results of CAM and Matrigel plug assay demonstrated that shikonin and sh-L can decrease neovascularization. Effect of shikonin was more obvious than sh-L at the same concentration. The results showed that sh-L decreased the toxicity, the rate of inhibition of migration and angiogenic suppression. The cellular uptake of the sh-L could be pictured because of the self-fluorescence. The self-fluorescence will be useful for conducting further research. Sh-L might be an excellent preparation for future clinical application to cancer patients.