Methotrexate-Conjugated Therapeutic Carbon Dots with Intrinsic Anticancer Activity for Breast Cancer Cell Inhibition

Carbon dots (CDs), a type of zero-dimensional carbon-based nanoparticle that possesses a size less than 10 nm, have been extensively investigated for cancer therapy. However, in such a field, most of them function only as nanocarriers for drug delivery, lacking intrinsic anticancer activity. In this project, we have developed a Y15*-CDs-MTX conjugation system composed of therapeutic CDs primarily derived from Y15 (1,2,4,5-benzenetetramine tetrahydrochloride, a focal adhesion kinase inhibitor used for cancer therapy) and another therapeutic anticancer drug, methotrexate (MTX), for breast cancer cell inhibition. The optical, structural, and morphological characterizations reveal that Y15*-CDs exhibit excitation-dependent photoluminescence emission, possess a size of 5–6 nm, and contain abundant amine groups on the surface. Following conjugation with MTX via an amidation reaction, the sizes of the obtained Y15*-CDs-MTX are 7–10 nm with a reduced zeta potential. The content of MTX in Y15*-CDs-MTX was determined to be 80% by mass. In vitro studies on breast cancer cells (MDA-MB-231, MCF-7, and 4T1 cells) and healthy cells (HFF) demonstrate that Y15*-CDs possess cancer cell-selective inhibitory activity, showing greater cytotoxicity toward cancer cells than healthy cells. The Y15*-CDs-MTX system, which combines the anticancer effects of both Y15*-CDs and MTX with the drug delivery capability of Y15*-CDs, exhibits enhanced cytotoxicity against breast cancer cells compared with Y15*-CDs or MTX alone. In contrast, lower cytotoxicity was still observed in the HFF cells. According to the cellular distribution study using MDA-MB-231 and HFF cells as models, the fluorescence of Y15*-CDs-MTX observed in MDA-MB-231 was higher than that in HFF cells. It indicates that Y15*-CDs-MTX preferentially enter breast cancer cells over healthy cells, which can be another reason that explains the cancer cell-selective property of Y15*-CDs-MTX. Therefore, the results in our work suggest the great potential of the developed Y15*-CDs-MTX system in breast cancer therapy.