Objective To investigate the antitumor effect of shikonin loaded milk derived exosomes on hepatoma cells.

Methods The experimental study was conducted. Exosomes were isolated from milk by differential centrifugation and be loaded by shikonin to constructed a nano drug loading system. The shikonin content of this nano drug loading system was determined and calculated by spectrophotometry. The cytotoxicity of shikonin loaded milk derived exosomes was evaluated by sulfonyl rhodamine B colorimetry and cell apoptosis was determined by annexin V-FITC/propidium iodide assay. Western blotting was used to detect the Bax and Bcl-2 protein expression level in hepatoma cells. Human HepG2 hepatoma cells treated with shikonin were set as the shikonin treated group and human HepG2 hepatoma cells treated with shikonin loaded milk derived exosomes were set as the shikonin loaded milk derived exosomes treated group. Observa-tion indictors: (1) the loading percentage of shikonin in shikonin loaded milk derived exosomes; (2) the cytotoxicity of shikonin loaded milk derived exosomes on human HepG2 hepatoma cells; (3) cell apoptosis of human HepG2 hepatoma cells; (4) Bax and Bcl-2 protein expression level in human HepG2 hepatoma cells. Measurement data with normal distribution were represented as Mean±SD, and comparison between groups were analyzed using the t test.

Results (1) The loading percentage of shikonin in shikonin loaded milk derived exosomes: the loading percentage of shikonin in shikonin loaded milk derived exosomes was 22.8%. (2) The cytotoxicity of shikonin loaded milk derived exosomes on human HepG2 hepatoma cells: the survival rates of hepatoma cells were 53.9%±2.9% and 45.4%±1.9% in the shikonin treated group and the shikonin loaded milk derived exosomes treated group, respectively, showing a significant difference (t=46.27, P<0.05). (3) Cell apoptosis of human HepG2 hepatoma cells: the early apoptosis rates of hepatoma cells were 11.3%±1.5% and 14.8%±2.2% in the shikonin treated group and the shikonin loaded milk derived exosomes treated group, respectively, showing no significant difference (t=1.37, P>0.05). The late and overall apoptosis rates of hepatoma cells were 32.3%±1.3% and 43.6%±4.3% in the shikonin treated group, versus 38.7%±3.2% and 53.5%±4.4% in the shikonin loaded milk derived exosomes treated group, showing significant differences (t=37.39, 30.97, P<0.05). (4) Bax and Bcl-2 protein expression level in human HepG2 hepatoma cells: Bax and Bcl-2 protein expre-ssion level were 232.0±2.6 and 32.0±1.6 in the shikonin treated group, versus 286.0±3.8 and 17.0±1.5 in the shikonin loaded milk derived exosomes treated group, showing significant differences (t=69.83, 53.32, P<0.05).

Conclusion The shikonin loaded milk derived exosomes have cytotoxic and apoptosis inducing effects on human HepG2 hepatoma cells, which can inhibit the growth of hepatoma cells.