Copper-cysteamine nanoparticle-mediated microwave dynamic therapy improves cancer treatment with induction of ferroptosis

Abstract

Photodynamic Therapy (PDT) holds a great promise for cancer patients, however, due to the hypoxic characteristics of most solid tumors and the limited penetration depth of light in tissues, the extensive clinical application of PDT is limited. Herein, we report microwave induced copper-cysteamine (Cu-Cy) nanoparticles-based PDT as a promising cancer treatment to overcome cancer resistance in combination with ferroptosis. The treatment efficiency of Cu-Cy-mediated microwave dynamic therapy (MWDT) tested on HCT15 colorectal cancer (CRC) cells via cell titer-blue cell viability assay and live/dead assay reveal that Cu-Cy upon MW irradiation can effectively destroy HCT15 CRC cells with average IC-50 values of 20 μg/mL. The cytotoxicity of Cu-Cy to tumor cells after MW stimulation can be alleviated by ferroptosis inhibitor. Furthermore, Cu-Cy mediated MWDT could deplete glutathione peroxide 4 (GPX4) and enhance lipid peroxides (LPO) and malondialdehyde (MDA). Our findings demonstrate that MW-activated Cu-Cy killed CRC cells by inducing ferroptosis. The superior in vivo antitumor efficacy of the Cu-Cy was corroborated by a HCT15 tumor-bearing mice model. Immunohistochemical experiments showed that the GPX4 expression level in Cu-Cy + MW group was significantly lower than that in other groups. Overall, these findings demonstrate that Cu-Cy nanoparticles have a safe and promising clinical application prospect in MWDT for deep-seated tumors and effectively inhibit tumor cell proliferation by inducing ferroptosis, which provides a potential solution for cancer resistance.

Keywords: Cell death; Colorectal cancer; Cu-Cy; Ferroptosis; Microwave; PDT.

Graphical abstract

Fig. 1

(A) UV–vis absorption curve of Cu-Cy nanoparticles in DI water. (B) The spectra of excitation (left, blue) at 607 nm and emission (right, red) at 365 nm of the Cu-Cy suspended in DI water. Inset: Images of the Cu-Cy at 1 mg/ml in DI water under UV light (left) and at ambient light (right).

Fig. 2

Detection of ROS in aqueous solution. (A) RNO-ID method was used to detect ROS produced by Cu-Cy aqueous solution (0.2 mg/ml) irradiated by MW of different power for 5 min. (B) RNO-ID method was used to detect ROS produced by Cu-Cy aqueous solution (0.2 mg/ml) irradiated with MW at 20 W for different time. (C) Fluorescence intensity of SOSG with the different Cu-Cy concentrations after MW irradiation. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

In vitro cell study of MWDT. (A) The cell viability of HCT15 cells was evaluated by cell titer blue cell viability assay after treating 0, 10, 20, 40, or 80 μg/mL of Cu-Cy nanoparticles with or without MW irradiation. (B) The live/dead staining images of HCT15 cells with Cu-Cy and MW irradiation (20 W, 3 min). (C) Colony formation study in HCT15 cells treated with MW. (D)The average number of clones was calculated. Scale bar: 50 μm ***P < 0.001 and ****P < 0.0001.

Fig. 4

Function of MWDT in ferroptosis induction. (A) Cell viability after treated with 5 mM of NAC, 100 μM of DFO, 1 μM of Fer-1, and 20 μg/mL of Cu-Cy under MW irradiation. (B) Western blot assay of GPX4 expression. (C) FCM assay of cellular LPO with BODIPY-C11 probe detection. (D) Relative fluorescence intensity of HCT15 stained by C11 BODIPY probe by FCM. (E) MDA examination under different treatments. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001.

Fig. 5

Antitumor effect of MWDT in mouse subcutaneous tumor models. (A) A schematic diagram of MWDT scheme for animal experiments. HCT15 tumor-bearing mice were treated with three injections of Cu-Cy on 0^th, 3rd, 6th, and 9th day. MW irradiation was performed on 1st, 4th, 7th, and 10th day. The tumor size and body weight were measured daily. (B) Images of each group at the end of the xenograft model experiment. (C) Tumor mass changes. (D) Body weight changes. (E) Tumor volume changes. (F) Histological observation of the tumor tissues with GPX4 staining. Scale bar: 50 μm *p < 0.05. (G) Immunoreactive scores (IRSs) were calculated and compared among groups.

Fig. 6

Schematic illustration of Cu-Cy mediated microwave dynamic therapy (MWDT) for colorectal cancer treatment by inducing cell ferroptosis.