Use of BMP9 in combination with NK cell and PD-L1 antibody in preparing medicament for treating liver cancer

A method for treating HBV-positive liver cancer comprising administering a BMP9 in combination with an NK cell and a PD-L1 antibody to a subject in need thereof, wherein

• the BMP9 is loaded on a drug-carrying microbubble (MB-BMP9);
• and
• the MB-BMP9 is prepared by the following steps:
• S1:
• dissolving 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol 2000 (PEG2000), and stearic acid-modified branched polyethylenimine-600 (Stearic-PEI600) in an organic solvent to obtain a first resulting mixture, and stirring the first resulting mixture for half an hour to give a phospholipid suspension;
• S2:
• uniformly mixing the phospholipid suspension, and removing the organic solvent from the phospholipid suspension to obtain a second resulting mixture;
• S3:
• adding phosphate-buffered saline (PBS) in the second resulting mixture to obtain a resulting solution, and incubating the resulting solution in a 60° C. water bath for 15 min to obtain an incubated solution;
• S4:
• shaking the incubated solution for 40 s in a bio-inert gas atmosphere, centrifuging the incubated solution to give an ultrasound microbubble, and washing the ultrasound microbubble to remove a phospholipid not forming the ultrasound microbubble;
• and
• S5:
• adding the BMP9 into a washed microbubble to obtain a third resulting mixture, and incubating the third resulting mixture for 1.5 h at room temperature to give the MB-BMP9;
• wherein
• in S1, a mass ratio of the DSPC, the DSPE-PEG2000, and the Stearic-PEI600 is 82:9:9;
• in S1 and S2, the organic solvent is a mixture of chloroform and methanol in a volume ratio of 9:1;
• in S4, the bio-inert gas atmosphere is perfluoropropane;
• in S4, a centrifugation condition is 400 g/min for 4 min;
• in S4, a method for washing is centrifugal floating;
• and
• in S5, an amount ratio of the BMP9 to the washed microbubble is 20 μg:108.