Intracellular Mechanistic Understanding of 2D MoS2 Nanosheets for Anti-Exocytosis-Enhanced Synergistic Cancer Therapy

Abstract

Emerging two-dimensional (2D) nanomaterials, such as transition-metal dichalcogenide (TMD) nanosheets (NSs), have shown tremendous potential for use in a wide variety of fields including cancer nanomedicine. The interaction of nanomaterials with biosystems is of critical importance for their safe and efficient application. However, a cellular-level understanding of the nano-bio interactions of these emerging 2D nanomaterials ( i. e., intracellular mechanisms) remains elusive. Here we chose molybdenum disulfide (MoS₂) NSs as representative 2D nanomaterials to gain a better understanding of their intracellular mechanisms of action in cancer cells, which play a significant role in both their fate and efficacy. MoS₂ NSs were found to be internalized through three pathways: clathrin → early endosomes → lysosomes, caveolae → early endosomes → lysosomes, and macropinocytosis → late endosomes → lysosomes. We also observed autophagy-mediated accumulation in the lysosomes and exocytosis-induced efflux of MoS₂ NSs. Based on these findings, we developed a strategy to achieve effective and synergistic in vivo cancer therapy with MoS₂ NSs loaded with low doses of drug through inhibiting exocytosis pathway-induced loss. To the best of our knowledge, this is the first systematic experimental report on the nano-bio interaction of 2D nanomaterials in cells and their application for anti-exocytosis-enhanced synergistic cancer therapy.

Keywords: MoS2; enhanced cancer therapy; intracellular trafficking network; nano-bio interactions; two-dimensional nanosheets.

Figure 1

Synthesis and characterization of MoS₂-based NSs. (a) Scheme of the exfoliation, PEGylation, and loading of MoS₂ NSs with therapeutic/diagnostic molecules. (b) Colloidal stability of (i) MoS₂ NSs and (ii) PEGylated MoS₂ NSs in water, PBS, and cell culture medium after 48 h incubation. (c) DLS hydrodynamic size distribution of PEGylated MoS₂ NSs. Inset: TEM image of PEGylated MoS₂ NSs. (d) AFM image of PEGylated MoS₂ NSs. The inset shows the thickness of PEGylated MoS₂ NSs. (e) Photothermal heating curves of pure water and MoS₂-PEG solutions with different concentrations (0.01, 0.05, and 0.1 mg/mL) under 808 nm laser irradiation at a power density of 1 W/cm². (f) UV–vis-NIR absorbance spectra of PEGylated MoS₂ NSs and PEGylated MoS₂/DOX NSs. (g) The released DOX percentage from fluorescent NSs at pH 7.4 and 5.0 after 2 or 3 h incubation.

Figure 2

Effect of energy on MoS₂-based NSs’ uptake. (a) The uptake of fluorescent MoS₂-based NSs (10 μg/mL) was tested by confocal laser scanning microscopy after 2 h incubation with HeLa and MCF-7 cells. The images in the top row are enlarged versions of the image area in the white box seen in the bottom row. Cell membrane was labeled with molecular probe (red). Scale bars: 10 μm. The uptake of fluorescent MoS₂-based NSs was measured by flow cytometer at indicated incubation times in (b) HeLa and (d) MCF-7 cells. Statistical analysis of intracellular fluorescence in (c) HeLa and (e) MCF-7 cells. Cells were pretreated with or without the metabolic inhibitor sodium azide (0.1%) and bafilomycin A (0.05 μM) for 30 min. Then cells were incubated with fluorescent MoS₂-based NSs for 2 h. Cytoplasmic fluorescence was measured by flow cytometer in (f) HeLa and (g) MCF-7 cells.

Figure 3

MoS₂-based NSs enter cells through clathrin-dependent, caveolin-dependent, and macropinocytosis pathways. Confocal images of (a) EGFP-clathrin-transfected, (b) EGFP-caveolae-transfected, and (c) EGFP-Rab34-transfected Hela cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. (d) Scatterplot of red and green pixel intensities of the cells shown in (a–c). Confocal images of (e) EGFP-clathrin-transfected, (f) EGFP-caveolae-transfected, and (g) EGFP-Rab34-transfected MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. (h) Scatterplot of red and green pixel intensities of the cells shown in (e–g). Scale bars: 10 μm.

Figure 4

MoS₂-based NSs accumulate in lysosomes. Schematic representation of the routes through which they enter the cell and accumulate in lysosomes in (a) HeLa and (b) MCF-7 cells. Confocal images of EGFP-Rab5-transfected (c) HeLa and (d) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. Confocal images of EGFP-Rab7-transfected (e) HeLa and (f) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. Confocal images of (g) HeLa and (h) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. The immunofluorescence experiment was performed to detect lysosomes with the primary antibody against Lamp1. Scale bars: 10 μm.

Figure 5

Autophagy involves the accumulation of MoS₂-based NSs in lysosomes. Confocal images of EGFP-LC3 transfected (a) HeLa and (b) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL). Confocal images of EGFP-LC3-transfected (c) HeLa and (d) MCF-7 cells incubated with PEGylated MoS₂ NSs (10 μg/mL) for 24 h and then treated with Lyso-Tracker Red probes for 30 min. (e) Statistical analysis of the numbers of co-localized vesicles between LC3 and fluorescent MoS₂-based NSs per cell. (Green = >3 co-localized vesicles; red = ≤3 co-localized vesicles). (f) Statistical analysis of the number of autophagosomes per cell after treatment. TEM images of (g) HeLa and (h) MCF-7 cells incubated with PEGylated MoS₂ NSs without loading content. Autophagosomes are formed after the same treatment. Triangles (Δ) indicate autophagosomes. Scale bars: 10 μm.

Figure 6

Exocytosis of MoS₂-based NSs. Confocal images of EGFP-Rab3-transfected (a) HeLa and (b) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. Confocal images of EGFP-Rab26-transfected (c) HeLa and (d) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. Confocal images of EGFP-Rab8-transfected (e) HeLa and (f) MCF-7 cells incubated with fluorescent MoS₂-based NSs for 2 h. Confocal images of EGFP-Rab10-transfected (g) HeLa and (h) MCF-7 cells incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 2 h. Scale bars: 10 μm.

Figure 7

Inhibition of exocytosis increases the accumulation of MoS₂-based NSs. Schematic representation of exocytosis pathways in (a) HeLa and (b) MCF-7 cells. (c) HeLa and (d) MCF-7 cells were pretreated with Exo1 for 2 h, and then cells were incubated with fluorescent MoS₂-based NSs (10 μg/mL) for 3 h. Cytoplasmic fluorescence was measured by flow cytometer. (e) HeLa and (f) MCF-7 cells were incubated in the presence or absence of Exo1 for 2 h after fluorescent MoS₂-based NS treatment. After that, we renewed the culture medium with fresh DMEM and incubated the cells for 3 h. Cytoplasmic fluorescence was then measured by flow cytometer.

Figure 8

(a) Schematic diagram illustrating the intracellular fates of MoS₂-based NSs. The process of trafficking starts with the internalization of NSs through macropinocytosis and both clathrin- and caveolae-dependent endocytosis, and then NSs are transported to early endosomes, shortly after that to late endosomes, and finally to lysosomes; autophagy is involved in the delivery of NSs to lysosomes; exocytosis participates in the secretion of NSs out of the cells; Exo1 inhibits the exocytosis pathways, which leads to the accumulation of MoS₂-based NSs in cells. Cell viabilities of (b) HeLa and (c) MCF-7 cells after various treatments indicated: (1) PEGylated MoS₂ NSs, (2) DOX, (3) PEGylated MoS₂/DOX NSs, (4) PEGylated MoS₂/DOX NSs + Exo1, (5) PEGylated MoS₂ NSs + NIR, (6) PEGylated MoS₂ NSs + NIR + Exo1, (7) PEGylated MoS₂/DOX NSs + NIR, and (8) PEGylated MoS₂/DOX NSs + NIR + Exo1 for 1, 2, or 3 h ([DOX] = 50 μg/mL, [MoS₂] = 50.2 μg/mL, [Exo1] = 50 μg/mL), respectively. NIR: 808 nm laser at a low power density of 0.4 W/cm² for 5 min after different incubation times. Exo1: pretreated with the cells for 2 h before the different incubation times. Afterward, cells were washed with PBS and incubated for another 24 h in fresh medium before the MTT assay.

Figure 9

Schematic representation of different therapeutic effects (a) without or (b) with the inhibition of Exo1. Although the same level of heat was generated due to the inhibition of Exo1, more PEGylated MoS₂ NSs accumulated in the cancer cells, and the gentle heat generated by these NSs directly damaged the organelles and proteins inside the cells. However, without the inhibition of Exo1, a certain number of the NSs were expelled from the cells. Because of the protection of the cell membranes and cellular homeostasis, the gentle heat generated by these NSs did not directly affect the organelles and proteins inside the cells.

Figure 10

MoS₂-based NSs for an in vivo triple-combination therapy with low dose. Treatment 1: Saline; Treatment 2: Exo1; Treatment 3: DOX; Treatment 4: PEGylated MoS₂/DOX NSs; Treatment 5: PEGylated MoS₂/DOX NSs + Exo1; Treatment 6: PEGylated MoS₂/DOX NSs + NIR; Treatment 7: PEGylated MoS₂/DOX NSs + NIR + Exo1. The doses of PEGylated MoS₂, DOX and Exo1 were 4.01, 4, and 4 mg/kg, respectively. NIR treatment was conducted by irradiating under an 808 nm laser (0.4 W/cm²) for 15 min at the tumor sites. (a) Photothermal images of MCF-7 tumor-bearing mice with different treatments were recorded by an IR thermal camera. (b) Temperature changes at tumor sites were monitored by the IR thermal camera during laser irradiation. (c) Tumor volume growth curves after various treatments for the different groups. (d) Body weight of MCF-7 tumor-bearing mice was measured every other day post treatment (n = 5, *** p < 0.001, ** p < 0.01, * p < 0.05). (e) H&E-stained histological images of tissue sections from heart, liver, spleen, lung, and kidney after 1 month of treatment with PEGylated MoS₂/DOX NSs together with Exo1. Saline was applied as control.