ROS Generative Black Phosphorus-Tamoxifen Nanosheets for Targeted Endocrine-Sonodynamic Synergistic Breast Cancer Therapy

Abstract

Introduction: Tamoxifen (TAM) has proven to be a therapeutic breakthrough to reduce mortality and recurrence in estrogen receptor-positive (ER+) breast cancer patients. However, the application of TAM exhibits low bioavailability, off-target toxicity, instinct and acquired TAM resistance.

Methods: We utilized black phosphorus (BP) as a drug carrier and sonosensitizer, integrated with TAM and tumor-targeting ligand folic acid (FA) to construct TAM@BP-FA for synergistic endocrine and sonodynamic therapy (SDT) of breast cancer. The exfoliated BP nanosheets were modified through in situ polymerization of dopamine, followed by electrostatic adsorption of TAM and FA. The anticancer effect of TAM@BP-FA was evaluated through in vitro cytotoxicity and in vivo antitumor model. RNA-sequencing (RNA-seq), quantitative real-time PCR, Western blot analysis, flow cytometry analysis and peripheral blood mononuclear cells (PBMCs) analysis were performed for mechanism investigation.

Results: TAM@BP-FA had satisfactory drug loading capacity, the TAM release behavior can be controlled through pH microenvironment and ultrasonic stimulation. An amount of hydroxyl radical (∙OH) and singlet oxygen (¹O₂) were as expected generated under ultrasound stimulation. TAM@BP-FA nanoplatform showed excellent internalization in both TAM-sensitive MCF7 and TAM-resistant (TMR) cells. Using TMR cells, TAM@BP-FA displayed significantly enhanced antitumor ability in comparison with TAM (7.7% vs 69.6% viability at 5μg/mL), the additional SDT further caused 15% more cell death. RNA-seq unraveled the TAM@BP-FA antitumor mechanisms including effects on cell cycle, apoptosis and cell proliferation. Further analysis showed additional SDT successfully triggering reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) reduction. Moreover, PBMCs exposed to TAM@BP-FA induced an antitumor immune response by natural killer (NK) cell upregulation and immunosuppression macrophage reduction.

Conclusion: The novel BP-based strategy not only delivers TAM specifically to tumor cells but also exhibits satisfactory antitumor effects through targeted therapy, SDT, and immune cell modulation. The nanoplatform may provide a superior synergistic strategy for breast cancer therapy.

Keywords: PBMC; black phosphorus; breast cancer; combination therapy; sonodynamic therapy; tamoxifen.

Scheme 1

(A) Preparation of the designed nanocomposites and (B) tumor accumulation after intravenous injection of nanocomposites. (C) Synergistic sonodynamic/hormone therapy, molecule mechanisms and potentiating affection on immune cells.

Figure 1

Synthesis and characterization of TAM@BP-FA nanocarriers. (A) TEM of TAM@BP-FA nanocomposites. (B) Fourier transform infrared spectroscopy (FTIR) of various nanomaterials. (C) UV-vis absorption of various nanomaterials. (D) X-ray powder diffraction (XRD) and (E) Raman spectra of bulk BP, BP and BP-PDA. (F–I) XPS survey spectra of TAM@BP-FA, high-resolution C 1s spectra, and high-resolution P 2p XPS spectra, high-resolution O 1s XPS spectra of TAM@BP-FA respectively.

Figure 2

In vitro drug release behavior. (A) Drug loading capacity and loading efficiency evaluation and (B) TAM release after treatment with ultrasound (1.5 W/cm2, 3 min).

Figure 3

In vitro SDT. (A) Illustration of the detection of ROS with specific probes. (B) Detection of singlet oxygen (¹O₂) using ABDA as a specific probe after application of ultrasound at various powers and (C) the corresponding quantitative analysis of fluorescence intensity after ultrasound. (D) Detection of ¹O₂ using ABDA as a specific probe with various nanomaterials and (E) the corresponding quantitative analysis of fluorescence intensity after ultrasound. (F) Detection of hydroxyl radicals (•OH) using PTA as a specific selective probe after application of ultrasound at various powers and (G) the corresponding quantitative analysis of fluorescence intensity after ultrasound. (H) Detection of •OH using PTA as a specific selective probe with various nanomaterials and (I) the corresponding quantitative analysis of fluorescence intensity. The corresponding mass concentrations of PDA, BP-PEG and BP-FA tested to detect nanomaterial-dependent ROS production were 100 μg/mL.

Figure 4

Cytotoxicity assays and cellular internalization. Cell viability of (A) MCF7 and (B) TMR cells exposed to free TAM or TAM@BP-FA at different concentrations. (C) Biotoxicity of BPNPs in MCF7 and TMR cells treated with doses from 0–100 μg/mL. (D) Quantification of fluorescence intensity in (E). (E) Fluorescence imaging of TMR cells incubated with Rho labeled TAM@BP-FA and TAM@BP, Nuclei were stained with DAPI. Data are means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Mechanism underlying the enhanced antitumor effects of TAM@BP-FA (A) Heatmap of the gene expression in TMR treated with free TAM and TAM@BP-FA, based on RNA-seq data. (B) Genes enriched in related pathways. (C) KEGG enrichment analysis of the top 10 key pathways unique to TAM@BP-FA. (D) The gene expression of CCND1 and MYC in TMR cells treated with free TAM or TAM@BP-FA. (E) Protein analysis of TMR cells after the above treatments (n = 2). (F and G) Cell cycle analysis of TMR with above treatments. Data are means ± SD (n = 3). *p < 0.05, ***p < 0.001 and ****p < 0.0001.

Figure 6

In vitro SDT study. (A) Relative cell viabilities of MCF7 after incubation with TAM@BP-FA with or without US irradiation for 24h (1.0 MHz, 1.5 W/cm2, 10 min, 50% duty cycle). (B) Relative cell viabilities of TMR after incubation with TAM@BP-FA with or without US irradiation for 24h (1.0 MHz, 1.5 W/cm2, 10 min, 50% duty cycle). (C) Fluorescence microscope images of calcein-AM (green, live cells) and PI (red, dead cells) staining, DCFDA staining (green, ROS detection), JC-1 staining (MMP detection) in TMR cells in the above-mentioned groups. Data are means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 7

In vitro immune responses. Gating strategy was provided in Figure S11A. (A) Relative changes in NK cells Macrophages and Macrophages type 2 were analyzed following PBMCs co-culture with medium from various materials and TMR for 48h. (B) Graphic representation of distributions and numbers of above-mentioned immune cells among the cohorts. Data are means ± SD (n = 3). ns no significance, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 8

Biodistribution of BPNPs in TMR tumor-bearing BALB/c nude mice. (A) In vivo distribution after intravenous injection of PBS, ICG and ICG@BP-FA. (B) Ex vivo distribution in main organs and tumors at 72 h post-injection of PBS, ICG and ICG@BP-FA. (C) Quantitative analysis of fluorescence intensity in the main organs and tumors. Data are means ± SD (n = 3).

Figure 9

In vivo synergistic antitumor effect (A) Treatment illustration. (B) Tumor growth curves in various groups with various treatments. (C) Tumor weight and digital photos of tumors from various groups after sacrifice (D) H&E staining and Ki67 staining of tumor tissues of various groups. G1-G7 represent mice treated with PBS, TAM, BP+US, BP-FA+US, TAM@BP+US, TAM@BP-FA and TAM@BP-FA+US respectively.