Glutathione Depletion-Induced ROS/NO Generation for Cascade Breast Cancer Therapy and Enhanced Anti-Tumor Immune Response

Abstract

Introduction: As an effective alternative choice to traditional mono-therapy, multifunctional nanoplatforms hold great promise for cancer therapy. Based on the strategies of Fenton-like reactions and reactive oxygen species (ROS)-mediated therapy, black phosphorus (BP) nanoplatform BP@Cu₂O@L-Arg (BCL) co-assembly of cuprous oxide (Cu₂O) and L-Arginine (L-Arg) nanoparticles was developed and evaluated for synergistic cascade breast cancer therapy.

Methods: Cu₂O particles were generated in situ on the surface of the BP nanosheets, followed by L-Arg incorporation through electrostatic interactions. In vitro ROS/nitric oxide (NO) generation and glutathione (GSH) depletion were evaluated. In vitro and in vivo anti-cancer activity were also assessed. Finally, immune response of BCL under ultrasound was investigated.

Results: Cu₂O was incorporated into BP to exhaust the overexpressed intracellular GSH in cancer cells via the Fenton reaction, thereby decreasing ROS consumption. Apart from being used as biocompatible carriers, BP nanoparticles served as sonosensitizers to produce excessive ROS under ultrasound irradiation. The enhanced ROS accumulation accelerated the oxidation of L-Arg, which further promoted NO generation for gas therapy. In vitro experiments revealed the outstanding therapeutic killing effects of BCL under ultrasound via mechanisms involving GSH deletion and excessive ROS and NO generation. In vivo studies have illustrated that the nanocomplex modified the immune response by promoting macrophage and CD8+ cell infiltration and inhibiting MDSC infiltration.

Discussion: BCL nanoparticles exhibited multifunctional characteristics for GSH depletion-induced ROS/NO generation, making a new multitherapy strategy for cascade breast cancer therapy.

Keywords: black phosphorus; chemodynamic therapy; combination cancer therapy; nitric oxide gas therapy; sonodynamic therapy.

Scheme 1

Schematic illustration of BCL composites synthesis procedure and the anti-cancer mechanisms including BP-based ROS generation, GSH deletion, NO production, enhanced anti-cancer immune response.

Figure 1

Characterization of nanoparticles (A) TEM image of BP NSs; (B) TEM and (C) HRTEM images of BCL; (D) Raman spectrum of BP NSs; (E) XRD patterns of BP NSs and BC; (F) Zeta potentials of BP, BC and BCL; (G) FT-IR spectrum of L-Arg, BP, BC and BCL; (H) SEM image and elemental mapping of BCL. C(red), N(yellow), O(green), P(blue) and Cu(purple).

Figure 2

In vitro Cu release, ROS and NO production behaviors. (A) Time-dependent ¹O₂ generation by BC detected by a ABDA probe under US irradiation; (B) Relative F/F₀ at 428 nm according to (A); (C) Time-dependent ·OH generation by BC using a PTA probe under US irradiation; (D) Relative F/F₀ at 430 nm according to (C); (E) The accumulated releases of Cu from BCL at pH 7.4 and 5.5; (F) UV–vis spectra of TMB incubated with single H₂O₂ or/and BC at pH 7.4 and 5.0; (G) UV–vis spectra of TMB incubated with H₂O₂ and BC with pH 5.0 at different time points; (H) Relative F/F₀ at 370 nm according to (F); (I) GSH consumption curve at different time points; (J) GSH consumption curve incubated with BP or BC; (K) NO concentration of different groups with/without US irradiation for 3 min; (L) The specific reaction processes. All the US irradiation treatments were proceeded under 1.0 MHz and 1.5 W/cm² and treatment was 3 min.

Figure 3

In vitro toxicity of BCL nanocomplex. (A) Cell viability of 4T1 cells treated with increasing dosage of BCL (0–100μg/mL), with or without US irradiation; (B) Cell viability of 4T1 cells treated with same amount of L-Arg, BC, BCL (40μg/mL) with or without US irradiation; (C) GSH content in cells treated with PBS, BP, BC, BCL, BCL+US; (D) Representative CLSM images of intracellular ROS in 4T1 cells in above groups; (E) Representative CLSM images of NO generation in 4T1 in above groups (Scale bars, 100 μm); FACS analysis of ROS (F) and NO (G) generation in 4T1 in above groups. *p< 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

Figure 4

In vivo anti-tumor activity. (A) Time schedule of the treatment plan; (B) Representative tumor photographs of different mouse groups with locally injection of indicated treatments; (C) Tumor weight in above groups; (D)Tumor growth curves in above groups; (E) Body weight changes in above groups. Data are shown as mean ± SD (n = 3). **p < 0.01, ***p < 0.001. ****p < 0.0001.

Figure 5

Bioinformatics analysis revealed the immune-regulation effects of BCL plus SDT therapy. (A) Volcano plots of the differentially expressed genes in 4T1 cells treated with PBS and BCL+US; (B) The top KEGG enrichment terms and (C) GSEA analyses of these genes enriched in (A).

Figure 6

The in vivo immune responses. Populations of macrophages (CD45+/CD11b+/F4/80+) and MDSC (CD45+/CD11b+/Gr-1+) in (A) control group and (B) BCL+US group; Relative changes in B cells (CD45+/CD19+), CD4+T cells(CD45+/CD3+/CD4+) and CD8+T cells (CD45+/CD3+/CD8+) in (C) control group and (D) BCL+US group.