Screening of Cu4O3 NPs efficacy and its anticancer potential against cervical cancer

Cu4 O3 is the least explored copper oxide, and its nanoformulation is anticipated to have important therapeutic potential especially against cancer. The current study aimed to biosynthesize Cu4 O3 nanoparticles (NPs) using an aqueous extract of pumpkin seeds and evaluate its antiproliferative efficacy against cervical cells after screening on different cancer cell lines. The obtained NPs were characterized by different spectroscopic analyses, such as UV-vis, thermogravimetric, energy dispersive X-ray, and Fourier-transform infrared spectroscopy (FTIR). In addition, high-resolution transmission electron microscopes (HR-TEM) were used to observe the morphology of the biosynthesized NPs. The UV-vis spectra showed a peak at around 332 nm, confirming the formation of Cu4 O3 NPs. Moreover, FTIR and TAG analyses identified the presence of various bioactive phytoconstituents that might have worked as capping and stabilization agents and comparative stable NPs at very high temperatures, respectively. The HR-TEM data showed the spherical shape of Cu4 O3 NPs in the range of 100 nm. The Cu4 O3 NPs was screened on three different cancer cell lines viz., Hela, MDA-MB-231, and HCT-116 using cytotoxicity (MTT) reduction assay. In addition, Vero was taken as a normal epithelial (control) cell. The high responsive cell line in terms of least IC50 was further assessed for its anticancer potential using a battery of biological tests, including morphological alterations, induction of apoptosis/ROS generation, regulation of mitochondrial membrane potential (MMP), and suppression of cell adhesion/migration. Vero cells (control) showed a slight decline in % cell viability even at the highest tested Cu4 O3 NPs concentration. However, all the studied cancer cells viz., MDA-MB-231, HCT 116, and HeLa cells showed a dose-dependent decline in cell viability after the treatment with Cu4 O3 NPs with a calculated IC50 value of 10, 11, and 7.2 µg/mL, respectively. Based on the above data, Hela cells were chosen for further studies, that showed induction of apoptosis from 3.5 to 9-folds by three different staining techniques acridine orange/ethidium bromide (AO/EB), 4',6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI). The enhanced production of reactive oxygen species (>3.5-fold), modulation in MMP, and suppression of cell adhesion/migration were observed in the cells treated with Cu4 O3 NPs. The current study obtained the significant antiproliferative potential of Cu4 O3 NPs against the cervical cancer cell line, which needs to be confirmed further in a suitable in vivo model. Based on our results, we also recommend the green-based, eco-friendly, and cost-effective alternative method for synthesizing novel nanoformulation.