Ce6-modified Fe ions-doped carbon dots as multifunctional nanoplatform for ferroptosis and photodynamic synergistic therapy of melanoma

Abstract

Background: Despite the higher sensitivity of melanoma towards ferroptosis and photodynamic therapy (PDT), the lack of efficient ferroptosis inducers and the poor solubility of photosensitizers restrict their synergistic strategies. With unique advantages, carbon dots (CDs) are expected to serve as innovative building blocks for combination therapy of cancers.

Results: Herein, an ferroptosis/PDT integrated nanoplatform for melanoma therapy is constructed based on chlorin e6-modified Fe ions-doped carbon dots (Fe-CDs@Ce6). As a novel type of iron-carbon hybrid nanoparticles, the as-prepared Fe-CDs can selectively activate ferroptosis, prevent angiogenesis and inhibit the migration of mouse skin melanoma cells (B16), but have no toxicity to normal cells. The nano-conjugated structures facilitate not only the aqueous dispersibility of Ce6, but also the self-accumulation ability of Fe-CDs@Ce6 within melanoma area without requiring extra targets. Moreover, the therapeutic effects of Fe-CDs@Ce6 are synergistically enhanced due to the increased GSH depletion by PDT and the elevated singlet oxygen (¹O₂) production efficiency by Fe-CDs. When combined with laser irradiation, the tumor growth can be significantly suppressed by Fe-CDs@Ce6 through cyclic administration. The T₂-weighted magnetic resonance imaging (MRI) capability of Fe-CDs@Ce6 also reveals their potentials for cancer diagnosis and navigation therapy.

Conclusions: Our findings indicate the multifunctionality of Fe-CDs@Ce6 in effectively combining ferroptosis/PDT therapy, tumor targeting and MRI imaging, which enables Fe-CDs@Ce6 to become promising biocompatible nanoplatform for the treatment of melanoma.

Keywords: Carbon dots; Ce6; Fe ions-doping; Ferroptosis; Melanoma; Photodynamic therapy.

Scheme 1

Schematic illustration of the synthesis procedure of Ce6-modified Fe ions-doped carbon dots (Fe-CDs@Ce6) as a multifunctional nanoplatform for ferroptosis/photodynamic synergistic melanoma therapy

Fig. 1

Characterization of Fe-CDs and Fe-CDs@Ce6. A Schematic illustration of the synthetic route of Fe-CDs and Fe-CDs-Ce6. B TEM image of Fe-CDs (inset: HR-TEM image and histogram of size distribution of Fe-CDs). C Maximum excitation and emission PL spectra of Fe-CDs (inset: photographs of Fe-CDs solution under daylight (left) and 365 nm UV light irradiation (right)). D XPS spectra of Fe-CDs. E Comparison of deconvoluted Fe 2p XPS spectra of GlcFe and Fe-CDs. F-G TEM images of Fe-CDs@Ce6 at low magnification and high magnification. H Comparison of deconvoluted N 1 s XPS spectra of Fe-CDs and Fe-CDs@Ce6. I UV–Vis absorption spectra of Fe-CDs and Fe-CDs@Ce6 (inset: photographs of (a) Ce6 and (b) Fe-CDs@Ce6 in PBS solution under daylight (left) and 365 nm UV light irradiation (right)). J PL spectra of Fe-CDs@Ce6 at different excitation wavelengths

Fig. 2

In vitro assay of cytotoxicity and oxidative stress. A Cell viability was assessed by CCK-8 assay. B GSH content detection. C The detection of singlet oxygen in the solution. D Detection of reactive oxygen species (ROS) levels in human umbilical vein endothelial cells co-cultured with B16 cell culture medium. Scale bar: 500 μm. E ROS levels. F Detecting changes in mitochondrial membrane potential in human umbilical vein endothelial cells co-cultured with B16 cell culture medium. Scale bar: 100 μm. G Quantification of the ratio of red signal (JC1 aggregates) to green signal (JC1 monomers), normalised to the intensity of the signal in non-treated control cells. (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered statistically significant)

Fig. 3

Evaluation of the ability to inhibit the proliferation of B16 cells. A B16 cells were treated with Fe-CDs and Fe-CDs@Ce6. Microscopy images were captured after staining with the LIVE/DEAD kit. Live cells are stained with green and dead cells with red (n = 3). Scale bar: 100 μm. B Cells were subjected to pulse-labeling with EdU as specified. Green fluorescence represents the EDU positive cells, and blue fluorescence represents the Hoechst stained cells. (n = 3). Scale bar: 600 μm. C Quantitative cell viability of the Live/Dead assay images. D Cell apoptosis of PBS, Ce6, Fe-CDs, Fe-CDs@Ce6, Fe-CDs@Ce6 + PDT by flow cytometry analysis. E Quantitative analyses of EdU assay. F Clone formation assay for assessing clone formation. Scale bar: 60 mm. G Histogram of cell clone formation rate. H The RNA expression levels of xCT, HO-1, and GPX4 (n = 4). I The protein expression levels of xCT, HO-1, and GPX4 (n = 3) (NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 4

Evaluation of the ability to inhibit the proliferation of B16 cells. A The invasion of B16 cells was assessed using a transwell assay (n = 3). Scale bar: 50 μm. B Data statistics of cell invasion in transwell chamber. C The comet assay was employed to detect DNA fragmentation in B16 cells (n = 3). Scale bar: 50 μm. D Fe-CDs can reduce the F-actin level in B16 cells. The green fluorescence represents F-actin, and the blue fluorescence represents DAPI. Scale bar: 80 µm, 80 µm, 80 µm and 20 µm from left to right. (n = 5). E The length of the comet tail was measured as the distance from the leading edge of the nucleus to the tails end. F F-actin levels were quantified using ImageJ software. G The RNA expression levels of β-catenin, Lef1, and Wnt3a (n = 4). H Protein expression levels of β-catenin, Lef1, and Wnt3a (n = 3) (NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 5

In vivo experiments of melanoma therapy. A The time schedule of this study. B Optical images of harvested tumors after the treatments (n = 6). Scale bar: 3 mm. C The volume of tumors from the mice in different groups (n = 6). D The weight of tumors from the mice in different groups (n = 6). E Tumor tissues were subjected to TUNEL staining following different treatments, with nuclei stained in blue and TUNEL-positive cells in green (n = 3). Scale bar: 100 μm. F Quantitative result of TUNEL assay was analyzed. G Quantitative result of hematoxylin–eosin staining was analyzed. H Staining of tumor tissues post-treatment using Hematoxylin and Eosin (H&E) (n = 3). Scale bar: 200 µm and 20 µm from top to bottom. (NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 6

Tumor-targeting ability, magnetic resonance imaging (MRI) ability and in vivo metabolism assay. A The B16 cells treated with Fe-CDs, when fixed and stained with Prussian blue, exhibited a blue color indicative of iron (n = 3). Scale bar: 200 μm and 50 μm from left to right. B Quantitative result of Prussian blue was analyzed. C The B16 cells treated with Fe-CDs were fixed and photographed using transmission electron microscopy. The right figure shows mitochondrial vacuolization. Scale bar: 2 μm and 1 μm from top to bottom. D The linear relationship of Fe-CDs@Ce6 solutions at different concentrations, and T₂-weighted MR images of Fe-CDs@Ce6 solutions at various concentrations. E In vivo T₂-weighted MRI and the relative signal intensity of the tumor following administration of Fe-CDs@Ce6. Scale bar: 50 mm. F The tail vein injection of Fe-CDs@Ce6 combined with IR800 in mice was performed to observe representative images of the distribution of Fe-CDs@Ce6 in the mouse body. Scale bar: 50 mm. G Representative fluorescence images of dissected organs at 30 min, 1, 24, 36, and 48 h after the injection of Fe-CDs@Ce6-IR800. From top to bottom: brain, heart, liver, lungs, and kidneys (n = 3, ****p < 0.0001)

Fig. 7

Analysis of mRNA expressions through transcriptome sequencing after combined therapeutic intervention. A The KEGG analysis revealed 20 pathways significantly enriched with upregulated differentially expressed genes. B The KEGG analysis revealed 20 pathways significantly enriched with downregulated differentially expressed genes. C Heat map diagram based on differentially expressed genes from the control and test groups. D GSEA enrichment plots of differentially expressed genes centralized in ferroptosis signaling pathway. E GSEA enrichment plots of differentially expressed genes centralized in melanogenesis signaling pathway. F The tissue sections obtained from treated mice were subjected to immunohistochemical staining for the proteins β-catenin, Lef1, HO-1, and GPX4. Scale bar: 50 μm. G The expression levels of β-catenin, xCT, Lef1, HO-1, and GPX4 proteins were assessed in the tissues obtained from mice after treatment. H A diagram illustrating the proposed mechanism