Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 May 10:16:3241-3254.
doi: 10.2147/IJN.S305857. eCollection 2021.

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 Zhao  1
Affiliations

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

Yandi Tan et al. Int J Nanomedicine. .

Abstract

Purpose: 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: drug delivery; immune checkpoint inhibitors; nanomaterials; tumor therapy; ultrasonics.

PubMed Disclaimer

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Characterization of sPD-1/Ce6-NBs. (A) Schematic diagram of sPD-1/Ce6-NBs. (B) Diameter and the mean particle size of sPD-1/Ce6-NBs. (C) Scanning electron microscopy of sPD-1/Ce6-NBs (magnification, x50,000). (D) Zeta potential of sPD-1/Ce6-NBs. (E) Red fluorescence of sPD-1 on the surface of sPD-1-NBs under a laser scanning confocal microscope (magnification, x10000). (F) Red fluorescence of Ce6 on the surface of sPD-1/Ce6-NBs under a fluorescence microscope (magnification, x200). (G) Gel electrophoresis showing the DNA loading capacity of sPD-1/Ce6-NBs. (H) UV-vis absorption spectra of sPD-1/Ce6-NBs and free Ce6, which showed no variation.
Figure 2
Figure 2
Pharmacokinetic study of sPD-1/Ce6-NBs. The tumor tissue of the sPD-1/Ce6-NBs group had more Ce6 accumulated in terms of time and concentration than that of the Ce6 group. (A) In vivo FL imaging of mice after intravenous injection of Ce6 and sPD-1/Ce6-NBs. (B) Semi-quantitative analysis of the FL signal of the tumor region in panel (A). (C) Ex vivo FL imaging of the heart, liver, spleen, lung, kidney and tumors at 24 h post-injection. (D) Quantitative analysis of the various organs shown in panel (C). (E) Images of frozen tumor sections at 24 h post-injection (magnification, x400). (F) Semi-quantification of the fluorescence intensity of Ce6 shown in panel. (*P <0.05, **P<0.01 versus the Ce6 group).
Figure 3
Figure 3
Tumor inhibition rate after in vivo treatment. (A) Schematic illustration showing the treatment process of sPD-1/Ce6-NBs. The same volume of NBs or PBS was administered to the animals through tail vein injection every 3 days (five times in total). With the exception of the control group, tumors were exposed to ultrasound irradiation immediately after injection, and the tumors of the Ce6, Ce6-NBs and SPD-1/Ce6-NBs groups were exposed to ultrasound irradiation again at 6 h post-injection. The curves of tumor growth shown in panel (B) indicated that all treatments effectively delayed tumor growth in varying degrees. The sPD-1/Ce6-NBs groups exhibited a marked therapeutic effect. The body weight of mice at different times shown in panel (C) and the liver index of mice in the different groups shown in panel (D) indicated that sPD-1/Ce6-NBs were no toxic in tumor.
Figure 4
Figure 4
Analysis of apoptosis of tumor tissues, cells, protein and mRNA. (A) Histological examination by hematoxylin and eosin staining (magnification, x200). All treatments led to tissue apoptosis in varying degrees, with the SPD-1/Ce6-NBs group being the most obvious. (B) TUNEL images of tumor tissues (magnification, x200). (C) Semi-quantification of the fluorescence intensity shown in panel (B). The trend of cell apoptosis was the same as that of tissues. (D) Immunochemistry staining images of Bax and Bcl-2 (magnification, x200). (E) Protein expression of Bax and Bcl-2. The expression of Bax protein in all the treatment groups was higher than that in the control groups, with the SPD-1/Ce6-NBs group being the most obvious. The opposite results were obtained for Bcl-2. (F) The mRNA expression of Bax and Bcl-2 followed the same trend as that of the protein. (*P <0.05, **P<0.01 versus the control group).
Figure 5
Figure 5
Antitumor mechanism of sPD-1-mediated immunotherapy and Ce6-assisted sonodynamic therapy. (A) Immunofluorescence images of PD-L1 (magnification, x200). (B) Immunochemistry images of CRT (magnification, x200). (C) Semi-quantification of the fluorescence intensity shown in panel (A). Compared with the control group, the protein and mRNA expression of PD-L1 shown in panels (C) and (D) was significantly lower in the SPD-1-NBs and SPD-1/Ce6-NBs groups. (E) The opposite results were obtained for the gene of sPD-1. The protein expression of CRT shown in panel (F) was significantly higher in the Ce6, Ce6-NB and SPD-1/Ce6-NBs groups. (*P <0.05, **P<0.01 versus the control group).
Figure 6
Figure 6
Immune activity enhanced by sPD-1/Ce6-NBs in vivo. The cytotoxic activity of natural killer splenic lymphocytes in panel (A) and cytotoxic T cell splenic lymphocytes in panel (B) was 2 and 20-fold stronger, respectively, than that of the control group. (C) The mRNA expression levels of CD86, CD80, IFN-γ, TNF-α and IL-2 in the treatment groups were higher than those of the control group, which indicated that sPD-1/Ce6-NBs had good immune activity. (*P <0.05, **P<0.01 versus the control group).
See this image and copyright information in PMC

References

    1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424. doi: 10.3322/caac.21492 - DOI - PubMed
    1. Chen Q, Chen M, Liu Z. Local biomaterials-assisted cancer immunotherapy to trigger systemic antitumor responses. Chem Soc Rev. 2019;48(22):5506–5526. doi: 10.1039/c9cs00271e - DOI - PubMed
    1. Li ZW, Li B, Peng D, et al. Expression and clinical significance of PD1 in hepatocellular carcinoma tissues detected by a novel mouse anti-human PD1 monoclonal antibody. Int J Oncol. 2018;52(6):2079–2092. doi: 10.3892/ijo.2018.4358 - DOI - PMC - PubMed
    1. Feng Z, Rong P, Wang W. Meta-analysis of the efficacy and safety of PD-1/PD-L1 inhibitors administered alone or in combination with anti-VEGF agents in advanced hepatocellular carcinoma. Gut. 2020;69(10):1904–1906. doi: 10.1136/gutjnl-2019-320116 - DOI - PubMed
    1. Cheng AL, Hsu C, Chan SL, Choo SP, Kudo M. Challenges of combination therapy with immune checkpoint inhibitors for hepatocellular carcinoma. J Hepatol. 2020;72(2):307–319. doi: 10.1016/j.jhep.2019.09.025 - DOI - PubMed

MeSH terms