Combination therapy has been considered one of the most promising approaches for improving the therapeutic effects of anticancer drugs. This is the first study that uses two different antioxidants in full-characterized niosomal formulation and thoroughly evaluates their synergistic effects on breast cancer cells. In this study, in-silico studies of hydrophilic and hydrophobic drugs (ascorbic acid: Asc and curcumin: Cur) interactions and release were investigated and validated by a set of in vitro experiments to reveal the significant improvement in breast cancer therapy using a co-delivery approach by niosomal nanocarrier. The niosomal nanoparticles containing surfactants (Span 60 and Tween 60) and cholesterol at 2:1 M ratio were prepared through the film hydration method. A systematic evaluation of nanoniosomes was carried out. The release profile demonstrated two phases (initial burst followed by sustained release) and a pH-dependent release schedule over 72 h. The optimized niosomal preparation displayed superior storage stability for up to 2 months at 4 °C, exhibiting extremely minor changes in pharmaceutical encapsulation efficiency and size. Free dual drugs (Asc + Cur) and dual-drug loaded niosomes (Niosomal (Asc + Cur)) enhanced the apoptotic activity and cytotoxicity and inhibited cell migration which confirmed the synergistic effect of co-encapsulated drugs. Also, significant up-regulation of p53 and Bax genes was observed in cells treated with Asc + Cur and Niosomal (Asc + Cur), while the anti-apoptotic Bcl-2 gene was down-regulated. These results were in correlation with the increase in the enzyme activity of SOD, CAT, and caspase, and the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) upon treatment with the mentioned drugs. Furthermore, these anti-cancer effects were higher when using Niosomal (Asc + Cur) than Asc + Cur. Histopathological examination also revealed that Niosomal (Asc + Cur) had a lower mitosis index, invasion, and pleomorphism than Asc + Cur. These findings indicated that niosomal formulation for co-delivery of Asc and Cur would offer a promising delivery system for an effective breast cancer treatment.
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