Carbon Nanodots Loaded with Natural Bioactive Compounds for P-gp Inhibition, Antimetastatic Activity, and Biosensing in 2D and 3D Breast Cancer Cells

Breast cancer is one of the most common cancers in women and has a major impact on cancer-related deaths worldwide. The combination therapy of drugs based on nanomaterials has emerged as an effective approach toward the different cancer treatments. While carbon nanodots (CDs) are mainly utilized for bioimaging, their potential as innovative nanocarriers remains largely untapped. Therefore, in this study, we synthesized biomass-derived carbon nanodots, and their formulation was optimized using Response Surface Methodology (RSM). The optimized CDs were coloaded with curcumin (Cur) and quercetin (Quer) (coloaded carbon nanodots) as model natural bioactive compounds to evaluate their synergistic therapeutic effect on breast cancer. The stability of the synthesized CDs was tested at various pH values (4-8.5) and over time, revealing excellent stability for up to 120 h even under acidic or basic conditions. UV-vis spectroscopy, XRD, Zeta potential, and FTIR confirmed the loading of these bioactive molecules on carbon nanodots. It has also been found that loading of curcumin and quercetin on carbon nanodots increases the stability and bioavailability of curcumin. Antiproliferative and antimetastatic properties of the coloaded carbon nanodots were further studied on the MCF-7 breast cancer cell line. The data represent that these coloaded carbon nanodots exhibit strong cytotoxicity and antimigration effects against the MCF-7. Notably, coloaded carbon nanodots significantly enhanced the apoptosis, ROS production, nucleus degeneration, and tumor inhibition <i>in vitro</i> in comparison to single bioactive-loaded carbon nanodots. The enhancement occurs because the coloaded carbon nanodots block multidrug resistance and the metastasis of cancer cells by downregulating the expression of membrane-bound P-glycoprotein (P-gp), resulting in the accumulation of the curcumin in the cancer cells. Additionally, they also downregulate the BIRC gene expression, which is the biomarker for triple negative breast cancer cells, whereas they enhance the expression of the p53 gene. To mimic the <i>in vivo</i> tumor microenvironment and address ethical concerns associated with animal studies, the penetration and efficacy of the coloaded CDs were also evaluated in 3D spheroid models of breast cancer. These models demonstrated efficient penetration and therapeutic action of the coloaded CDs, highlighting their translational potential for future cancer therapy.