CD44 Receptor-Mediated Ferroptosis Induction by Hyaluronic Acid Carbon Quantum Dots in Triple-Negative Breast Cancer Cells Through Downregulation of SLC7A11 Pathway

Abstract

The field of cancer therapy is actively pursuing highly effective self-targeted drug delivery materials endowed with exceptional properties. Recently, hyaluronic acid (HA), a naturally occurring polysaccharide, has been recognized as a potential target ligand for CD44 receptors, which are frequently expressed on various solid tumor cells targeted in cancer therapy. HA carbon quantum dots (CQDs) exhibit several advantageous properties, including a high surface area-to-volume ratio, small particle size, biocompatibility, and low cytotoxicity, making them ideal for biomedical applications, such as CD44-targeted drug delivery in ferroptosis-based cancer therapy. In this study, we synthesized HA-CQDs to enhance CD44-mediated ligand-receptor interactions targeting triple-negative breast cancer (TNBC). CQDs facilitate the intracellular generation of reactive oxygen species (ROS), leading to glutathione depletion. These processes result in crucial actions such as the downregulation of glutathione peroxidase 4, downregulation of solute carrier family 7 member 11, and inhibition of cystine intake. The subsequent intracellular ROS, originating from lipid peroxidation, induces ferroptosis. Our HA-CQDs engage CD44 receptors, selectively targeting TNBCs and enhancing cancer recognition. This interaction potentially enhances the nanoplatform-based CD44 targeted therapeutic effects in inducing ferroptosis.

Keywords: carbon quantum dots (CQDs); ferroptosis; glutathione depletion; hyaluronic acid (HA); lipid peroxidation; reactive oxygen species (ROS).

Figure 1

Schematic diagram illustrating HA-CQD-mediated ferroptosis-based CD44-targeted anticancer activity in triple-negative breast cancer cells.

Figure 2

Synthesis procedure of hyaluronic acid carbon quantum dots.

Figure 3

Characterization of HA-CQDs. (a,b) TEM and SEAD patterns of HA-CQDs, respectively; (c) BET analysis of HA-CQDs; (d) Micro Raman spectra of HA-CQDs; (e) H-1 NMR spectra; and (f) TGA analysis of HA and HA-CQDs. The red line indicates the thermal decomposition of the HA sample, which occurs in two distinct stages.

Figure 4

(a) FTIR spectra of HA and HA-CQDs; XPS spectra of HA-CQDs depict (a) wide scan, (b) C (1s) state, (c) C (1s) state, (d) N (1s) state, and (e) O (1s) state.

Figure 5

Docking analysis of (a) HA, (b) HA-CQDs, demonstrating their interaction with the 3D structure of the CD44 receptor, and identifying the best-docked sites of (c) HA, (d) HA-CQDs in the CD44 receptor, and (e) HA-CQDs based on the Ferroptosis anticancer mechanism.

Figure 6

In vitro anticancer effect of HA-CQDs. Cell viability of (a) MDA-MB-231 (b) HepG2, and (c) Fibroblast cells when treated with various concentrations of HA-CQDs for 24 h using WST-1 assay. (*: statistical difference compared to 0 µg/mL, n.s.: non-statistical differences).

Figure 7

Intracellular ROS measurement. ROS generation was determined using the DCFH-DA probe. Fluorescence intensity and fluorescence images of DCFH-DA after exposure to HA-CQDs (0, 50, and 100 μg/mL) for 6 h in MDA-MB-231 cells (a,b) and (c,d) HepG2 cells. (II) The ratio of GSH/GSSG includes (e) the mechanism of GSH depletion, (f) the ratio in MDA-MB-231 cells, and (g) the ratio in HepG2 cells treated with 0, 50, and 100 μg/mL of HA-CQDs for 24 h. (*: statistical difference compared to 0 µg/mL, n.s.: non-statistical differences).

Figure 8

(a) Provides insights into mRNA expression of ferroptosis-related genes. The mRNA expressions of (b) SLC7A11, (c) GPX4, and (d) ACSL4 were measured in MDA-MB-231 cells treated with HA-CQDs; and expressions of (e) SLC7A11, (f) GPX4, (g) ACSL4 were evaluated in HepG2 cells treated with HA-CQDs. GAPDH was utilized as a housekeeping gene. * indicates a significant difference with the control (p < 0.05). A ferroptosis mechanism based on lipid peroxidation featured (h) MDA levels in (i) MDA-MB-231 group and (j) HepG2 group after treatment with HA-CQDs at concentrations of 50 and 100 μg/mL for 24 h.