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. 2023 Jun;10(16):e2206707.
doi: 10.1002/advs.202206707. Epub 2023 Apr 17.

DNA Damage Inducer Mitoxantrone Amplifies Synergistic Mild-Photothermal Chemotherapy for TNBC via Decreasing Heat Shock Protein 70 Expression

Zuqin Chen  1   2 , Sunfan Li  3 , Fangzhou Li  4 , Cheng Qin  1 , Xianlei Li  4 , Guangchao Qing  4 , Jinjin Wang  4 , Bozhang Xia  4 , Fuxue Zhang  4 , Liangliang Meng  5 , Xing-Jie Liang  4 , Yueyong Xiao  2
Affiliations

DNA Damage Inducer Mitoxantrone Amplifies Synergistic Mild-Photothermal Chemotherapy for TNBC via Decreasing Heat Shock Protein 70 Expression

Zuqin Chen et al. Adv Sci (Weinh). 2023 Jun.

Abstract

Patients with triple-negative breast cancer (TNBC) have the worst clinical outcomes when compared to other subtypes of breast cancer. Nanotechnology-assisted photothermal therapy (PTT) opens new opportunities for precise cancer treatment. However, thermoresistance caused by PTT, as well as uncertainty in the physiological metabolism of existing phototherapeutic nanoformulations, severely limit their clinical applications. Herein, based on the clinically chemotherapeutic drug mitoxantrone (MTO), a multifunctional nanoplatform (MTO-micelles) is developed to realize mutually synergistic mild-photothermal chemotherapy. MTO with excellent near-infrared absorption (≈669 nm) can function not only as a chemotherapeutic agent but also as a photothermal transduction agent with elevated photothermal conversion efficacy (ƞ = 54.62%). MTO-micelles can accumulate at the tumor site through the enhanced permeability and retention effect. Following local near-infrared irradiation, mild hyperthermia (<50 °C) assists MTO in binding tumor cell DNA, resulting in chemotherapeutic sensitization. In addition, downregulation of heat shock protein 70 (HSP70) expression due to enhanced DNA damage can in turn weaken tumor thermoresistance, boosting the efficacy of mild PTT. Both in vitro and in vivo studies indicate that MTO-micelles possess excellent synergetic tumor inhibition effects. Therefore, the mild-photothermal chemotherapy strategy based on MTO-micelles has a promising prospect in the clinical transformation of TNBC treatment.

Keywords: DNA damage; chemotherapy; heat shock protein; mild-photothermal therapy; mitoxantrone; polymeric micelle; triple-negative breast cancer (TNBC).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Schematic illustration of the multifunctional MTO‐micelles for mutually synergistic mild‐photothermal chemotherapy of TNBC.
Figure 1
Figure 1
Characterizations of MTO‐micelles. A) The TEM image and B) DLS measurement of MTO‐micelles. Scale bar: 100 nm. C) UV–vis absorption spectra of free MTO and MTO‐micelles in aqueous and methanol solution (10 µg mL−1 of equivalent MTO concentration), respectively. D) Photothermal heating curves of PBS and MTO‐micelles (80 µg mL−1 of equivalent MTO concentration) under 665 nm laser irradiation (0.6 W cm−2). E) Photothermal heating curves of MTO‐micelles at various concentrations (20, 40, and 80 µg mL−1) under 665 nm laser irradiation (0.6 W cm−2). F) Photothermal heating curves of MTO‐micelles (60 µg mL−1) under 665‐nm irradiation under various laser intensities (0.2, 0.4, and 0.6 W cm−2). G) Photothermal stability of MTO‐micelles (100 µg mL−1 of equivalent MTO concentration) exposed to a 665‐nm laser (0.6 W cm−2). H) Photothermal heating/cooling curves of MTO‐micelles aqueous solution (100 µg mL−1 of equivalent MTO concentration, 1 mL) in the laser‐on and off state. ΔT stands for the temperature increasement of the solution. I) The time constant for MTO‐micelles heat transformation (τ) was calculated by plotting linear time data from the system cooling period against the negative natural logarithm of the system driving force temperature.
Figure 2
Figure 2
Cellular uptake of MTO‐micelles. A) Representative CLSM photographs and B) quantitative fluorescence analysis of 4T1 cells following incubation with free MTO or MTO‐micelles (1.0 µg mL−1 of MTO‐equivalent concentration) at various time intervals. The nucleus was stained with DAPI (blue; ex: 358 nm, em: 461 nm), and MTO moieties exhibited red channel fluorescence emission (ex: 633 nm, em: 680 nm). Data are presented as the mean ± SD (n = 3) (*P < 0.05 and ****P < 0.0001). C) Quantitative fluorescence analysis of 4T1 cells incubated for 4 h with various concentrations of MTO‐equivalent. Data are presented as the mean ± SD (n = 3) (*P < 0.05, **P < 0.01, and ****P < 0.0001). D) Flow cytometric measurement of 4T1 cells incubated for 4 h with free MTO or MTO‐micelles (1.0 µg mL−1 of MTO‐equivalent concentration).
Figure 3
Figure 3
In vitro γH2AX foci formation and HSP70 expression evaluation. A) Representative CLSM images and B) quantitative analysis of γH2AX foci formation in 4T1 cells of different treatment groups. Data shown are mean ± SD (n = 20) (*P < 0.05, **P < 0.01). C) Representative CLSM images and D) quantitative analysis of HSP70 expression in 4T1 cells of different treatment groups. Data shown are mean ± SD (n = 10) (****P < 0.0001). E) Western blots of γH2AX and HSP70 with β‐Actin as the internal reference, and F) Relative expression levels of γH2AX and HSP70 in 4T1 cells of different treatment groups (MTO concentration was 1.0 µg mL−1, under 24 h of incubation). Data shown are mean ± SD (n = 3) (*P < 0.05, **P < 0.01, ****P < 0.0001).
Figure 4
Figure 4
In vitro antitumor efficiency of MTO‐micelles. A) In vitro viability of 4T1 cells treated with laser (665 nm, 0.6 W cm−2, 2 min), MTO‐free, MTO‐free + laser (665 nm, 0.6 W cm−2, 2 min), MTO‐micelles, and MTO‐micelles + laser (665 nm, 0.6 W cm−2, 2 min) by CCK‐8 assay. Data shown are mean ± SD (n = 3) (*P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001). B) Flow cytometry analysis of 4T1 cells apoptosis induced by different treatments. MTO equivalent concentration was 1.0 µg mL−1. The incubation time was 24 h. Annexin‐positive cells were regarded as apoptotic cells. C) Apoptotic rate of different treatment groups determined by Annexin V‐FITC/PI staining. Data shown are mean ± SD (n = 3) (*P < 0.05, **P < 0.01). D) Representative images and E) quantitative analysis of the colonies of different treatment groups. Data shown are mean ± SD (n = 3) (*P < 0.05).
Figure 5
Figure 5
Intratumoral accumulation of MTO‐micelles and photothermal effect In vivo. A) Fluorescence images and B) mean fluorescence intensity of 4T1 tumor‐bearing mice at various time points following administration of MTO·2HCl or MTO‐micelles (5 mg kg−1 of equivalent MTO). Tumor sites are denoted by red dashed line circles. Values shown are mean ± SD (n = 3) (**P < 0.01). C) Ex vivo fluorescence images of major organs and tumors obtained 24 h after administration. D) HPLC analysis of MTO content in mice tumor tissues following intravenous administration of MTO·2HCl or MTO‐micelles (5 mg kg−1 of equivalent MTO). Values shown are mean ± SD (n = 3) (**P < 0.01, ***P < 0.001). E) Representative in vivo thermal images of 4T1 tumor‐bearing mice, and F) intratumoral temperature curves following intravenous injections of saline, MTO·2HCl, or MTO‐micelles (5 mg kg−1 of equivalent MTO) under NIR irradiation (665 nm, 0.6 W cm−2). Values shown are mean ± SD (n = 3) (****compared with saline + laser, P < 0.0001; #### compared with MTO‐free + laser, P < 0.0001).
Figure 6
Figure 6
In vivo mild‐photothermal chemotherapy and histopathological analysis of tumor tissues. A) Tumor growth curves of mice during therapy. Values shown are mean ± SD (n = 5) (****P < 0.0001). B) The photographs of the extracted tumors at day 18 following various treatments. C) Average weights of tumors at day 18 following various treatments. Values shown are mean ± SD (n = 5) (****P < 0.0001). D) Representative H&E and IHC (Ki‐67, cleaved caspase‐3, γH2AX, and HSP70) staining images of mice tumor tissues at the end of treatment. Scale bar: 100 µm.
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