Breast cancer, a leading cause of cancer-related deaths in women, faces significant treatment challenges due to drug resistance. Methotrexate (MTX), an effective chemotherapy medication for various malignancies, often encounters resistance in breast cancer, reducing its efficacy. This resistance underscores the urgent need for novel therapeutic strategies. Nano-drug delivery systems (NDDSs), such as niosomes, offer a promising solution. These systems can encapsulate both hydrophobic and hydrophilic drugs, enabling reduced dosages and enhanced delivery. By overcoming drug resistance, NDDSs pave the way for more effective combination chemotherapy in breast cancer treatment.

In this study, two pharmacological agents, (i) methotrexate (MTX) as a hydrophilic drug and (ii) silibinin (SiL) as a hydrophobic drug, were simultaneously loaded into the hydrophilic and lipophilic part of niosome, respectively. Niosomes were synthesized by the thin film layer hydration method and characterized by zeta sizer, FTIR, and TEM. Also, MTT assay, DAPI, dead/alive and F-actin/DAPI staining, and spheroid cell culture were used to analyze nanoparticle biocompatibility, cell viability, apoptosis, cell adhesion density, and anti-tumor response, respectively, in 2D and 3D cultured MDA-MB-231 mammospheres. Additionally, an in-silico network analysis was conducted to investigate the interaction of MTX and SiL with human proteins, especially those that contribute to breast cancer pathways.

Ideal niosomes with spherical morphology, ~ 87 nm size and ~-15 mV zeta potential, and high biocompatibility were successfully synthesized. The combination of MTX and SiL exhibited significant synergistic effects, as evidenced by the Fa value of 0.5 for NiO@MTXSiL at a concentration of 3.84 µg/mL. This value is markedly lower compared to those observed for MTXSiL (11.78 µg/mL), SiL (26.24 µg/mL), and MTX (18.48 µg/mL). Importantly, in the TNBC microtumor model, lower doses of NiO@MTXSiL achieved an almost complete anti-tumor drug response, leaving only ~ 6% residual tumor cells. Moreover, our computational analysis identified seven human proteins (i.e. BRCA1, CCND1, CDK4, CDK6, CDKN1A, Rb1, and Tp53) as breast cancer key players in the MTX and SiL interaction network with human proteins. Of these, Tp53 emerges as the most crucial protein, serving as a hub-bottleneck node, a common direct neighbor of MTX and SiL, and a key player in four breast cancer subtypes.

The designed nano-niosome, NiO@MTXSiL, is safe, stable, and has an optimal size and surface charge. It offers high drug loading capacity for co-delivering hydrophobic and hydrophilic chemotherapeutics with different anti-cancer mechanisms, improving anti-tumor response and overcoming MDR. It shows higher cytotoxicity against MDA-MB-231 breast cancer cells compared to free drugs, making it a promising candidate to combat MTX resistance in breast cancer.