Nanobubbles Containing sPD-1 and Ce6 Mediate Combination Immunotherapy and Suppress Hepatocellular Carcinoma in Mice

Yandi Tan,1,* Shiqi Yang,1,* Yao Ma,2 Jinlin Li,1 Qian Xie,1 Chaoqi Liu,1 Yun Zhao1 1Medical College of China Three Gorges University, Yichang, Hubei, People’s Republic of China; 2Department of Ultrasonography, Yichang Central People’s Hospital, Yichang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yun Zhao; Chaoqi LiuMedical College of China Three Gorges University, Yichang, Hubei, 443000, People’s Republic of ChinaEmail zhaoyun@ctgu.edu.cn; ctgulcq@163.comPurpose: Immune checkpoint inhibitors (ICIs) and sonodynamic therapy (SDT) are types of immunotherapy. In order to combine soluble programmed cell death protein 1 (sPD-1)-mediated immune checkpoint therapy and chlorin e6 (Ce6)-assisted SDT, nanobubbles (NBs) were generated to simultaneously load sPD-1 and Ce6.Materials and Methods: The sPD-1/Ce6-NBs, which were prepared by thin-film hydration and mechanical oscillation, had a stable physical condition, and delivered sPD-1 and Ce6 in a targeted manner. NBs could strengthen tumor suppression by increasing tumor-targeting accumulation of Ce6 and sPD-1, and by inducing ultrasound-targeted NB destruction. A mouse H22 cell hepatoma xenograft model was used to evaluate the synergetic immunotherapeutic effect and mechanism of sPD-1/Ce6-NBs.Results: By observing the tumor inhibition rate, tissue and cell apoptosis, apoptosis-related genes and protein expression, the best immunotherapeutic effect was exhibited by the sPD-1/Ce6-NBs group. The immunotherapeutic mechanism initially demonstrated that when tumor cells were transfected by sPD-1 delivered by NBs, which downregulated the expression of programmed death-ligand 1 (PD-L1) in tumor cells, and blocked the PD-1/PD-L1 signaling pathway, which improved T-cell-mediated tumor inhibition. Furthermore, ICIs combined with SDT induced immunogenic cell death by translocating calreticulin to the cell surface and then synergistically enhancing antitumor immune responses.Conclusion: In conclusion, sPD-1/Ce6-NBs were successfully designed. Ultrasound-mediated sPD-1/Ce6-NBs are potentially effective delivery systems for combination immunotherapy of hepatocellular carcinoma.Keywords: immune checkpoint inhibitors, ultrasonics, drug delivery, tumor therapy, nanomaterials