Evaluation of ZnO nanoparticles as an adjuvant therapeutic substance in the radiotherapy of solid tumors using the chorioallantoic membrane assay

Abstract

Introduction: Innovative cancer therapies aim to exert selective cytotoxic effects while preventing tumor proliferation. Zinc oxide nanoparticles (ZnO-NP) have emerged as promising antitumor agents due to their cytotoxicity in various human cancer cell lines and favorable biocompatibility. This study explored the antineoplastic potential of ZnO-NPs alone and in combination with radiotherapy — a common cancer treatment hindered by tumor radioresistance and other side effects. Aims: We aimed to evaluate whether ZnO-NP enhance the efficacy of radiotherapy by reduction of cell viability and vascular structures within the tumor area using the Hen’s Egg Test – Chorioallantoic Membrane Model (HET-CAM-Assay) which included a xenograft of human hepatocellular carcinoma (Huh-7) cell-derived tumors. Methods: CAMs were divided into four groups: untreated, ZnO-NP only, radiation only (8 Gy), and combination treatment. Depending on the study group, ZnO-NP (13.5 µg) were injected intravascularly 120 hours after the xenografts were applied while radiation treatment was performed 24 hours later. After another 24 hours, tumors were excised and analyzed immunohistochemically for morphology (H&E, pan-keratin), apoptosis (Cleaved Caspase-3), proliferation (Ki-67), and angiogenesis (α-smooth muscle actin). Results: Radiation alone increased apoptosis and proliferation while reducing angiogenesis. ZnO-NP monotherapy showed similar, though milder effects. Both treatments elevated CC3 and Ki-67 levels, suggesting cytotoxicity accompanied by compensatory proliferation. Notably, the combination therapy increased apoptosis, while proliferation was reduced, and decreased vascularization — indicating synergistic effects. Conclusion: ZnO-NPs exhibit notable antineoplastic effects in ovo, particularly in combination with radiation treatment. The findings suggest that ZnO-NPs may enhance radiotherapeutic outcomes by promoting apoptosis, suppressing proliferation, and inhibiting angiogenesis. Their potential as radiosensitizers in future cancer therapies is to be evaluated further.