A minimalist and robust chemo-photothermal nanoplatform capable of augmenting autophagy-modulated immune response against breast cancer

Hui Ming¹, Bowen Li¹, Hailong Tian¹, Li Zhou¹, Jingwen Jiang¹, Tingting Zhang¹, Ling Qiao², Peijie Wu², Edouard C Nice³, Wei Zhang^{4

5}, Weifeng He⁶, Canhua Huang¹, Haiyuan Zhang⁷

Affiliations

¹ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, 610041, PR China.
² School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
³ Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia.
⁴ West China Biomedical Big Data Center, West China Hospital, Sichuan University, Chengdu, 610041, China.
⁵ Mental Health Center and Psychiatric Laboratory, The State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China.
⁶ Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burn and Combined Injury, Chongqing Key Laboratory for Disease Proteomics, Army Military Medical University, Chongqing, 400038, China.
⁷ School of Basic Medicine, Health Science Center, Yangtze University, Jingzhou, China.

PMID: 35634171
PMCID: PMC9130112
DOI: 10.1016/j.mtbio.2022.100289

A minimalist and robust chemo-photothermal nanoplatform capable of augmenting autophagy-modulated immune response against breast cancer

Hui Ming et al. Mater Today Bio. 2022.

. 2022 May 13:15:100289.

doi: 10.1016/j.mtbio.2022.100289. eCollection 2022 Jun.

Authors

Hui Ming¹, Bowen Li¹, Hailong Tian¹, Li Zhou¹, Jingwen Jiang¹, Tingting Zhang¹, Ling Qiao², Peijie Wu², Edouard C Nice³, Wei Zhang^{4

5}, Weifeng He⁶, Canhua Huang¹, Haiyuan Zhang⁷

Affiliations

¹ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, 610041, PR China.
² School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
³ Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia.
⁴ West China Biomedical Big Data Center, West China Hospital, Sichuan University, Chengdu, 610041, China.
⁵ Mental Health Center and Psychiatric Laboratory, The State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, China.
⁶ Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burn and Combined Injury, Chongqing Key Laboratory for Disease Proteomics, Army Military Medical University, Chongqing, 400038, China.
⁷ School of Basic Medicine, Health Science Center, Yangtze University, Jingzhou, China.

PMID: 35634171
PMCID: PMC9130112
DOI: 10.1016/j.mtbio.2022.100289

Abstract

Previously used in anti-fungal therapy, itraconazole has now been shown to be successful in treating advanced breast cancer (NCT00798135). However, its poor solubility still restricts its application in clinical treatment. There is therefore an urgent need for combined methods to enhance the therapeutic effect of itraconazole (IC) in breast cancer treatment. With this goal, co-assembled IC/IR820 NPs with synergistic photonic hyperthermia and itraconazole payloads have been constructed to overcome these shortcomings. The IC/IR820 NPs show an enhanced therapeutic effect on breast cancer by inducing reactive oxygen species (ROS)-mediated apoptosis and autophagic death. Further evaluation in a mouse model has shown impressive effects of the IC/IR820 NPs on both inhibiting tumor metastasis and activating immunity to prevent tumor recurrence. Mechanistically, itraconazole may promote both tumor cell antigen presentation through autophagy and the activation of dendritic cells to induce an immune response, which displays a synergistic effect with the immune response generated by photothermal therapy to inhibit tumor recurrence. This strategy of combining itraconazole and IR820 into one minimalist and robust nanoplatform through co-assembly results in excellent therapeutic efficacy, suggesting its potential application as an alternative method for the clinical treatment of breast cancer.

Keywords: Autophagy; Breast cancer; IR820; Immunotherapy; Itraconazole; Photothermal therapy.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

**Scheme 1**
The preparation of the IC/IR820 NPs allows amplified apoptosis, autophagy, and immunogenic cell death during breast cancer therapy. Created with BioRender.com.

**Fig. 1**
**Characterization and intercellular uptake of IC/IR820 NPs.** (A) UV–vis absorption spectra of IC/IR820 NPs. (B) TEM image of IC/IR820 NPs. Scale bar: 100 nm. (C) Size distribution of IC/IR820 NPs. (D) Photothermal activity of IC/IR820 NPs dispersed in water at various concentrations (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 5 min). (E) Infrared thermal images of IR820 and IC/IR820 NPs in water after laser irradiation (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 5 min). (F) Fluorescence microscopy images and (G) flow cytometric results of cell uptake of IC/IR820 NPs into MDA-MB-231 cells at different time points. Scale bar: 20 μm. (H) Fluorescence microscopy images and (I) flow cytometric results of cell uptake of IR820 and IC/IR820 nanoparticles in MDA-MB-231 cells at different concentrations after incubation for 4 h. Scale bar: 20 μm.

**Fig. 2**
**The anti-breast cancer effect of IC/IR820 NPs.** (A–C) Viability of MDA-MB-231, MCF-7, and 4T1 cells co-cultured with IC, IR820, and IC/IR820 nanoparticles. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 20 s). (D–G) Representative images for colony formation of MDA-MB-231, MCF-7, and 4T1 cells co-cultured with IC, IR820, and IC/IR820 nanoparticles. ∗∗∗P < 0.001. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 30 s). (H–K) Quantification of MDA-MB-231, MCF-7, and 4T1 cells co-cultured with IC, IR820, and IC/IR820 nanoparticles in the EDU assay. Scale bar: 20 μm. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 20 s). (L–N) LDH assay of MDA-MB-231, MCF-7, and 4T1 cells co-cultured with IC, IR820, and IC/IR820 nanoparticles. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 20 s).

**Fig. 3**
**IC/IR820 NPs-mediated ROS induce breast cancer cell death.** (A–C) Immunofluorescence images to show subcellular localization of IR820 and IC/IR820 NPs. Scale bar: 20 μm. (D) Fluorescence microscopy images and (E) flow cytometry analysis for intracellular ROS generation of MDA-MB-231 cells using DCFH-DA as a probe. Scale bar: 20 μm. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 2 min). (F–H) Flow cytometry statistical analysis for intracellular ROS generation of MDA-MB-231, MCF-7 and 4T1 cells using DCFH-DA as a probe. Scale bar: 20 μm. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 2 min). (I–K) Viability of specific groups with or without NAC treatment in MDA-MB-231, MCF-7 and 4T1 cells. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 20 s).

**Fig. 4**
**IC/IR820 NPs trigger ROS-dependent apoptosis in breast cancer cells.** (A–D) Apoptosis of IR820, IC, and IC/IR820 NPs were evaluated by annexin V-FITC/PI staining in MDA-MB-231, MCF-7 and 4T1 cells, quantification of apoptotic cell ratio. (λ = 808 nm, P = 1.0 W/cm²; the irradiation time = 2 min). (E–G) Immunoblot analysis of apoptotic markers for MDA-MB-231, MCF-7 and 4T1 cells treated IC, IR820, IC/IR820 with or without NAC treatment. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 2 min). (H–J) Viability of specific groups with or without ZVAD treatment in MDA-MB-231, MCF-7 and 4T1 cells. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 20 s).

**Fig. 5**
**IC/IR820 NPs-mediated ROS induce autophagic cell death in breast cancer cells.** (A–C) Immunoblot analysis of autophagic markers for MDA-MB-231, MCF-7 and 4T1 cells treated IC, IR820, IC/IR820 with or without NAC treatment. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 2 min). (D–G) Immunofluorescence assays display subcellular localization of LC3B puncta in MDA-MB-231 and MCF-7 cells treated IC/IR820 NPs with or without NAC treatment, quantification of LC3B puncta per cell. Scale bar: 10 μm. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 30s). (H–J) Immunofluorescence images to show subcellular localization of LC3-GFP and LC3-RFP. Scale bar: 20 μm. (K–M) Viability of specific groups with or without 3 MA treatment in MDA-MB-231, MCF-7 and 4T1 cells. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 20 s).

**Fig. 6**
The anti-breast cancer effect of IC/IR820 NPs *in vivo*. (A) *In vivo* distribution of IR820 and IC/IR820 NPs at different times. (B) Schematic illustration of the procedure of tumor treatment *in vivo*. Created with BioRender.com. (C) *In vivo* thermal imaging of different groups of treated mice. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 5 min). (D) Representative images of isolated tumors. Scale bar: 1 cm. (E) The volume of tumors from each group (5 mice per group) was measured at the indicated time points. (F) The body weight of mice in each group was measured at the indicated time points. (G) The weight of individual tumors and the inhibition rate. (H–L) Representative immunohistochemistry images of mice treated with vehicle, IR820, or IC/IR820 NPs intravenously through tail vein at the indicated time points, quantification of Ki67, 8-OHDG, cleaved-caspase 3, and LC3B expression score. Scale bar: 50 μm. (λ = 808 nm, P = 1.0 W/cm²; irradiation time = 5 min).

**Fig. 7**
**IC/IR820 NPs inhibit breast cancer metastasis.** (A–B) Immunoblot analysis of EMT markers for MDA-MB-231 and MCF-7 cells treated with IC, IR820, IC/IR820 with or without NAC treatment/irradiation (808 nm, 1.0 W cm⁻², 20 s). (C–E) Representative images of migration, invasion, and scratch test of MDA-MB-231 cells, quantification of migration/invasion cell number and migratory distance. Scale bar for migration and invasion assay: 50 μm. Scale bar for scratch test: 20 μm. (F–I) Representative lung, liver H&E staining of mice bearing 4T1 orthotopic breast tumor xenografts treated with vehicle, IR820 + Laser, IC, IC/IR820 NPs + Laser at the end of the treatment period. Quantification of the relative metastatic area in lungs and metastatic nodes in livers. Scale bar: 50 μm.

**Fig. 8**
**IC/IR820 NPs induce immune response and inhibit recurrence in mouse model.** (A) Schematic illustration of the treatment of bilateral tumors. Created with BioRender.com. (B) Distant tumor volumes. (C–E) Representative results of CD4⁺ T cells and CD8⁺ T cells. Quantification of percentage of CD4⁺ T cells and CD8⁺ T cells in the spleen. (F) Immunoblot analysis of E-cadherin, caspase3, cleaved-caspase3 for distal tumor treated with NS, IC, IR820, and IC/IR820 NPs. (G) Schematic illustration of the treatment of postsurgical mice. Created with BioRender.com. (H) Tumor volumes. (I–K) Representative results of CD4⁺ T cells and CD8⁺ T cells. Quantification of percentage of CD4⁺ T cells and CD8⁺ T cells in the spleen.

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A minimalist and robust chemo-photothermal nanoplatform capable of augmenting autophagy-modulated immune response against breast cancer

Affiliations

A minimalist and robust chemo-photothermal nanoplatform capable of augmenting autophagy-modulated immune response against breast cancer

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

LinkOut - more resources

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