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. 2021 Jul 25;11(17):8587-8604.
doi: 10.7150/thno.62572. eCollection 2021.

Mitochondria-targeted and ultrasound-responsive nanoparticles for oxygen and nitric oxide codelivery to reverse immunosuppression and enhance sonodynamic therapy for immune activation

Changwei Ji  1 , Jingxing Si  2   3 , Yan Xu  4 , Wenjing Zhang  4 , Yaqian Yang  4 , Xin He  1 , Hao Xu  4 , Xiaozhou Mou  2   3 , Hao Ren  4 , Hongqian Guo  1
Affiliations

Mitochondria-targeted and ultrasound-responsive nanoparticles for oxygen and nitric oxide codelivery to reverse immunosuppression and enhance sonodynamic therapy for immune activation

Changwei Ji et al. Theranostics. .

Abstract

Background: Sonodynamic therapy (SDT) is a promising strategy to inhibit tumor growth and activate antitumor immune responses for immunotherapy. However, the hypoxic and immunosuppressive tumor microenvironment limits its therapeutic efficacy and suppresses immune response. Methods: In this study, mitochondria-targeted and ultrasound-responsive nanoparticles were developed to co-deliver oxygen (O2) and nitric oxide (NO) to enhance SDT and immune response. This system (PIH-NO) was constructed with a human serum albumin-based NO donor (HSA-NO) to encapsulate perfluorodecalin (FDC) and the sonosensitizer (IR780). In vitro, the burst release of O2 and NO with US treatment to generate reactive oxygen species (ROS), the mitochondria targeting properties and mitochondrial dysfunction were evaluated in tumor cells. Moreover, in vivo, tumor accumulation, therapeutic efficacy, the immunosuppressive tumor microenvironment, immunogenic cell death, and immune activation after PIH-NO treatment were also studied in 4T1 tumor bearing mice. Results: PIH-NO could accumulate in the mitochondria and relive hypoxia. After US irradiation, O2 and NO displayed burst release to enhance SDT, generated strongly oxidizing peroxynitrite anions, and led to mitochondrial dysfunction. The release of NO increased blood perfusion and enhanced the accumulation of the formed nanoparticles. Owing to O2 and NO release with US, PIH-NO enhanced SDT to inhibit tumor growth and amplify immunogenic cell death in vitro and in vivo. Additionally, PIH-NO promoted the maturation of dendritic cells and increased the number of infiltrating immune cells. More importantly, PIH-NO polarized M2 macrophages into M1 phenotype and depleted myeloid-derived suppressor cells to reverse immunosuppression and enhance immune response. Conclusion: Our findings provide a simple strategy to co-deliver O2 and NO to enhance SDT and reverse immunosuppression, leading to an increase in the immune response for cancer immunotherapy.

Keywords: Immune response; Mitochondria-targeted; Oxygen and nitric oxide codelivery; Reverse immunosuppression; Sonodynamic therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Schematic illustration of PIH-NO for cancer therapy and immune activation. PIH-NO was constructed by IR780 and HSA-NO with oxygen saturation. After intravenous injection, it could increase blood perfusion, increase EPR effect and relive hypoxia. It could prefer to accumulate in mitochondria. After with ultrasound irradiation, it could release oxygen and NO to enhance SDT, and damage mitochondria, which induce immunogenic cell death to promote DC maturation. The generation NO and RNS could polarize M2 macrophages to M1 phenotype and reduce MDSC to reverse immunosuppression microenvironment. All these approaches increase CD8+ T cells infiltration to inhibit primary tumor and lung metastasis.
Figure 2
Figure 2
Characteristic of PIH-NO. (A) TEM images and (B) size distribution of PIH-NO. (C) The stability of PIH-NO within 48 h when stored at 4 °C and 37 °C. (D) UV-vis absorption spectra of IH and PIN-NO. (E) Time-dependent changes of dissolved oxygen concentrations in deoxygenated pure water after addition of water@O2 and PIH-NO@O2 with US treatment in the indicated period (6 min to 10 min). (F) The release of NO in PIH-NO (NO 60 μM) with or without US treatment. (G) The release of NO with different concentrations of PIH-NO (0, 20, and 60 μM). (H) The generation of singlet oxygen under US treatment determined by SOSG fluorescence. (I) Fluorescence spectrum of ONOO- in different groups evaluated by dihydrorhodamine 123 (DHR). Unless otherwise specified, US treatment is under 1.0 MHz and 1.0 W/cm2.
Figure 3
Figure 3
The cell viability of mitochondria-targeted PIH-NO in vitro. (A) Mitochondrial location of PIH-NO monitored by Mito-tracker fluorescence. Blue, green, and red represent DAPI, Mito-tracker Green, and IR780 fluorescence, respectively. The scale bar is 20 μm. (B) Cell viabilities of 4T1 cells after incubation with different groups at various doses. *p <0.05, **p <0.01 (PIH-NO with US vs. other treatments). (C) Calcein-AM (green)/PI (red) staining and (D) flow cytometry apoptosis of 4T1 cells after various treatments. The scale bar is 100 μm. Data are expressed as means ± SD (n = 3).
Figure 4
Figure 4
Intracellular ROS and NO generation. (A) Representative fluorescence images of hypoxia (red) in 4T1 tumor cells after different treatments. (B) ROS generation (DCFH-DA, green) and (C) Flow cytometry quantitative analysis of ROS generation in treated groups with or without US. The scale bar is 100 μm. (D) Representative fluorescence images and (E) semiquantitative analysis of NO release stained with DAF-FM (green) in 4T1 tumor cells after various treatments. The scale bar is 200 μm. *p <0.05 (PIH-NO without US vs. PIH without US), **p <0.01 (PIH-NO with US vs. other treatments). Data are expressed as means ± SD (n = 3).
Figure 5
Figure 5
Mitochondria destruction and ICD induced by PIH-NO. (A) Fluorescence images of lipid peroxide in 4T1 cells with different treatments detected by Liperfluo (green fluorescence) for the lipid peroxide-specific oxidation. The scale bar is 100 μm. (B) Mitochondrial membrane potential stained with JC-1 after various treatments. The scale bar is 100 μm. (C) CRT exposure on the surface of 4T1 cells. The scale bar is 100 μm. (D) Extracellular ATP and (E) HMGB-1 release from 4T1 tumor cells after various treatments. **p <0.01 (PIH-NO with US vs. other treatments). Data are expressed as means ± SD (n = 3).
Figure 6
Figure 6
Biodistribution of PIH-NO in vivo. (A) Real-time fluorescence images of 4T1 tumor-bearing mice after intravenous injection of PIH and PIH-NO (IR780, 78 μg/mL). (B) Ex vivo fluorescence images and (C) the corresponding relative mean fluorescence intensity of major organs and tumor at 36 h after PIH and PIH-NO treatments (**p <0.01, PIH-NO vs. PIH). (D) The blood perfusion detected by a Doppler flowmeter in the tumor after various treatments. (E) Immunofluorescence images of HIF-1α standing of tumor slices 24 h post injection of different formulations. The scale bar is 200 μm. Data are expressed as means ± SD (n = 3).
Figure 7
Figure 7
Inhibition of tumor and metastasis of PIH-NO in vivo. (A) Tumor growth curves and (B) average tumor weight after various treatments. (C) The number of lung metastasis. (D) H&E and TUNEL staining of tumor slices after various treatments on the 14th day. The scale bar is 200 μm. (E) H&E staining of lung metastatic nodules. The scale bar is 100 μm. *p <0.05, **p <0.01 (PIH-NO with US vs. other treatments). Data are expressed as means ± SD (n = 6).
Figure 8
Figure 8
Reverse immunosuppression of tumor microenvironment in vivo. (A, B) Flow cytometry analysis of M1 (CD11c+F4/80+CD86+) and M2 (CD11c+F4/80+CD206+) in treated tumors. (C, D) The proportion of M1 and M2 according to flow cytometry analysis. (E, F) Representative flow cytometry plots and percentages of MDSCs (CD11b+Gr-1+) in treated tumors. **p <0.01 (PIH-NO with US vs. other treatments). Data are expressed as means ± SD (n = 5).
Figure 9
Figure 9
Anti-tumor immune response after PIH-NO treatment. (A) CRT and HMGB-1 immunofluorescence staining of tumor slices after various treatments. The scale bar is 100 μm. (B, C) Representative flow cytometry plots and percentages of DCs (CD11b+CD86+CD80+) in tumor-draining lymph nodes for DC maturation in vivo. (D-F) Flow cytometry analysis, percentages, and immunofluorescence staining of CD8+ T cells (gated CD3+) in tumors. The scale bar is 100 μm. (G) TNF-α expression determined by ELISA. **p <0.01 (PIH-NO with US vs. other treatments). Data are expressed as means ± SD (n = 5).
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