Nanobubbles containing PD-L1 Ab and miR-424 mediated PD-L1 blockade, and its expression inhibition to enable and potentiate hepatocellular carcinoma immunotherapy in mice

Immune checkpoint inhibitors (ICI) therapy is the main type of immunotherapy for cancer. Current clinical trials are focused on enhancing anti-tumor effects through combinations of multiple ICIs with agents that cause tumor cell death and release tumor antigens. In this study, weprepared nanobubbles (NBs) to load programmed death-ligand 1 (PD-L1) antibody andmiR-424gene to evaluate the combined anti-tumor activity of the targeted NBs.The miR-424 gene was chosen to be an anti-tumor gene, which can target PD-L1 and Bcl-2, through bioinformatics analysis and target gene verification. Then, PD-L1 Ab/miR-424-NBs were prepared by thin-film hydration. The optimal shape, size, and character of the NBs were determined by scanning electron microscopy and Zeta potential study. In addition, the antibody binding rate and gene loading of the targeted NBs were studied by agarose gel electrophoresis and flow cytometry, respectively. The synergistic immunotherapeutic effect of anti-PD-L1 antibody andmiR-424in vivo and their mechanism were evaluated using an H22 hepatoma transplanted tumor model in mice,whichproved that the targeted NBs mediated the PD-L1 antibody toblock the PD-1/PD-L1 signaling pathway and the transfected miR-424gene to downregulate the PD-L1 expression of tumor cells, both of which enhanced the antitumor immune effect mediated by T cells. It was also found that the targeted NBs activated T cells, which released a large number of cytokines, such as IFN-γ and IL-2, to recruit and activate macrophages and NK cells. It is suggested that ultrasound-mediated PD-L1 antibody NBs delivering miR-424 can inhibit the growth of subcutaneously transplanted hepatocellular carcinoma in terms of apoptosis and immunity. Therefore, ultrasound-mediated targeted NBs are a potential effective carrier for liver cancer, and PD-L1 antibody and miR-424 have a synergistic anti-tumor immunotherapy effect.