Production of copper nanoparticules by the green chemistry method, loading of epigallocatechin combination on PLGA nano-carriers and investigation of their in vitro and in vivo anti-tumor effects on breast cancer

Although phytochemical compounds generally have antioxidant properties, they are used in cancer treatment because they show pro-oxidant effect due to Fenton reaction in the presence of free iron (Fe) and copper (Cu) ions. However, since the bioavailability of phytochemicals is low, their therapeutic doses are high and their selectivity is not at the desired level, making it difficult to use them in cancer treatment, studies to increase their bioadjectivity, increase their effectiveness at lower doses and develop targeted phytochemical treatment methods have recently gained importance. In recent studies, it has been shown that antioxidant phytochemicals in the presence of metallic free Cu and Fe have a pro-oxidant effect due to the redox reaction known as the Fenton reaction, and thus have the potential to kill cancer cells by DNA damage. In particular, the fact that Cu is at higher concentrations in cancer cells than normal cells increases the selectivity of phytochemicals on cancer cells. Therefore, by transporting the targeted phytochemical compounds together with Cu to the target tissue by nano-technological methods, it has been thought to increase the pro-oxidant activity of the phytochemical compound and to ensure that it is effective only in cancer tissue without damaging healthy tissues. Our aim is to transfer the epigallocatechin (EGC) to the target cancer tissue by loading the copper nanoparticles formed by green chemistry method with epigallocatechin, which is an important antioxidant phytochemical in green tea, to the target cancer tissue and to increase the anti-tumor activity. Bio-synthesized Cu nano-conjugates have long been found to be highly stable and biocompatible in in vitro and in vivo systems. While EGC was used as anti-oxidant phytochemical, copper was transformed into metallic nanoparticles by green chemistry method and both were loaded into poly (lactic-co-glycolic acid) (PLGA) nanomelles. For this purpose, the genotoxic, apoptotic and cytotoxic effects of different concentrations of nano-copper produced with green chemistry and EGC PLGA nano-particle on breast cancer cells (4T-1) will be analyzed and compared with intact breast cells. The IC50 of EGC was 2.89 mg ± 1.8757 in DMSO and PLGA-EGC-Cu within 72 hours, respectively; While it was 7.37 mg ± 12, 129, the IC50 of free nano copper was found to be 4.70 mg ± 0.3596. Epigallocatechin in PLGA required a higher concentration to cause death in 50 % of cells compared to epigallocatechin in DMSO. It also shows that the PLGA-EGC-Cu formulation kills 4T-1 cells within 24 hours, and living cells proliferate within 48 and 72 hours. The effective dose was determined in vitro, and then the same complex was given to experimental animals in vivo to investigate anti-tumor activity. In nude mice with 4T-1 tumors, PLGA-EGC-Cu combined treatment yielded the best results compared to free EGC and free nanofiber. Tumor volume was reduced approximately 15-fold, 3.5-fold, and 2.86-fold compared to the untreated group, nano Cu, PLGA-EGC-Cu, and PLGA-EGC treatment groups. Since the PLGA nanocarrier will be FITC labeled, the uptake of the nanoparticle applied to mice in the tumor could be followed by an IVIS imaging device. Thus, the pro-oxidant effect of the phytochemical was prevented from being effective on tumor cells at lower doses and damaging intact cells as it concentrated on cancer tissue. Keywords: Natural Antioxidants, Targeted Cancer Therapy, Copper, ROS, Pro-oxidant,CombinationTherapy,GreenChemistry