Green synthesis of copper oxide nanoparticles using walnut shell and their size dependent anticancer effects on breast and colorectal cancer cell lines

www.nature.com/scientificreports OPEN Green synthesis of copper oxide nanoparticles using walnut shell and their size dependent anticancer effects on breast and colorectal cancer cell lines Hanieh Abdollahzadeh 1, Yaghub Pazhang 1,2* , Asghar Zamani 3 & Yousef Sharafi 4 Metal oxide nanoparticles(NPs) contain unique properties which have made them attractive agents in cancer treatment. The CuO nanoparticles were green synthesized using walnut shell powder in different calcination temperatures (400°, 500°, 700°, and 900 °C). The CuO nanoparticles are characterized by FTIR, XRD, BET, SEM and DLS analyses. SEM and DLS analyses showed that by increasing the required calcination temperature for synthesizing the NPs, their size was increased. DPPH analysis displayed no significant anti-oxidative properties of the CuO NPs. The MTT analysis showed that all synthesized CuO NPs exhibited cytotoxic effects on MCF-7, HCT-116, and HEK-293 cell lines. Among the CuO NPs, the CuO-900 NPs showed the least cytotoxic effect on the HEK-293 cell line (IC50 = 330.8 µg/ml). Hoechst staining and real-time analysis suggested that the CuO-900 NPs induced apoptosis by elevation of p53 and Bax genes expression levels. Also, the CuO-900 NPs increased the Nrf-2 gene expression level in MCF-7 cells, despite the HCT-116 cells. As can be concluded from the results, the CuO-900 NPs exerted promising cytotoxic effects on breast and colon cancer cells. Keywords Walnut shell, Green synthesis, CuO nanoparticles, Cancer therapy, Apoptosis, p53, Nrf-2 Metal oxide nanoparticles have gained more attentions rather than other nanoparticles due to their unique properties and their applications in various fields, such as anti-microbial agents, in semi-conducting devices, textile industry, microelectronics, cosmeceutical products, and biomedical fi elds1. Because of the emerging anticancer effects of the metal oxide NPs, they have been considered as agents to remove cancer cells in preclinical studies2. In recent years, the synthesis of CuO nanoparticles has been the goal of scientific research because of their various application especially biomedical a pplications3–5. It has been reported that CuO nanoparticles are more cytotoxic to human cells rather than other metal oxide NPs6. However, the anti-cancer effects of CuO NPs were studied on many cancer types, including liver7, lung8, and breast9,10, cervical11 and pancreatic12 cancers. In 2023, 1,958,310 new cancer cases and 609,820 cancer deaths are estimated to occur in the United S tates13. Despite the existence of multiple approaches to treat cancer including chemotherapy, immunotherapy, radiotherapy, and surgery, not only the disease is still a main health challenge in worldwide but also its related deaths are increasing year by year13. Therefore, finding new agents and approaches to treat cancer is one of significant health challenges in the field of medical science. Based on the WHO report, three prevalent cancer types in women include breast, lung and colorectal cancers. On another hand, the most prevalent cancer types in the men are prostate, lung, and colorectal cancers14. Increase in cancer prevalence is related partly to the resistance of cancer cells to the conventional therapies. Beside the conventional therapeutic strategies, capabilities of nanoparticles as an agent to treat cancer and to effectively deliver anticancer drugs have made them as a hopeful approach for overcoming c ancer15. Numerous synthesis procedures have been developed to synthesize CuO nanoparticles, including co-precipitation, sol–gel technique, thermal decomposition, and hydrothermal methods. However, these techniques, in addition to the environmental impacts, are usually hard to produce on the industrial scale. Accordingly, more 1 Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran. 2Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran. 3Department of Nanotechnology, Faculty of Chemistry, Urmia University, Urmia, Iran. 4Dryland Agricultural Research Institute, Agricultural Research, Education and Extension Organization(AREEO), Maragheh, Iran. *email: Scientific Reports | (2024) 14:20323 | https://doi.org/10.1038/s41598-024-71234-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ attention has been recently paid to the green approach and biological method to the synthesis of nanoparticles, chiefly copper oxide n anoparticles16–19. Agricultural waste biomass particularly lignocellulosic wastes such as nut shells have increasingly attracted some attention as a low-cost renewable resource in various fi elds20,21. Walnut is always a popular fruit in the world. Based on incomplete statistics, thousands of tons of walnuts are consumed every year in the world and the shells are usually cast-off. More recently, we synthesized nanostructured magnesium oxide22, alumina23, and cerium oxide nanoparticles24 by walnut shell. This abundantly available agricultural waste is composed of cellulose, hemicellulose, and lignin5,25 which can act as fuel or sacrificial templates in preparing metal oxide nanostructures. In this protocol, walnut shell grind was mixed with aqueous solutions of metal nitrate in diverse ratios without using any toxic chemicals. Stirring followed by evaporation and calcination of paste, resulting in desired nanostructures. In this paper, we present similar process for the preparation of CuO nanoparticles with different sizes by using walnut shell powder. The aim of this study is the green synthesis of different dimensions of CuO NPs to investigate their anticancer effects. Material and methods General remarks Copper(II) nitrate hexahydrate, Cu(NO3)2.6H2O, from Merck and used without further purification. The walnut shell from a local walnut tree in Urmia (Iran) was crushed using a high-speed rotary cutting mill. X-ray diffraction patterns of the obtained materials were recorded at room temperature on Shimadzu XRD-6000 diffractometer with CuKα irradiation. The morphology of materials was observed by Hitachi S-4100 FESEM instrument (Japan). Also, BET analysis was performed by Belsorp-mini II-BEL, Inc. analyzer at 77 K. Fourier transform infrared (FT-IR) spectra were attained by a Bruker Vector 22 FT-IR spectrophotometer under ambient conditions in a KBr/Nujol mull in the range of 400–4000 cm−1. Copper oxide nanoparticles preparation Walnuts were purchased from local farmers and then the walnut shells separated from the kernel. Next, the walnut shells were grinded and the walnut shell powder was prepared. Copper oxide NPs was synthesized in the presence of walnut shell powder 30 g and copper(II) nitrate hexahydrate 6.65 g in 50 mL of deionized water (Millipore, Milli-Q grade). After 4 h stirring, the water was evaporated by a rotary evaporator under low pressure. The resulting paste was subsequently calcined at 400 °C for 4 h (at a heating rate of 10 °C/min) under open-air c (...truncated)