Hassan Amr, Al-Salmi Fawziah A, Abuamara Tamer M M, Matar Emadeldin R, Amer Mohamed E, Fayed Ebrahim M M, Hablas Mohamed G A, Mohammed Tahseen S, Ali Haytham E, Abd El-Fattah Fayez M, Abd Elhay Wagih M, Zoair Mohammad A, Mohamed Aly F, Sharaf Eman M, Dessoky Eldessoky S, Alharthi Fahad, Althagafi Hussam Awwadh E, Abd El Maksoud Ahmed I
Department of Bioinformatics, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat, Egypt.
Biology Department, College of Sciences, Taif University, Taif, Saudi Arabia.
Front Oncol. 2022 Oct 27;12:933750. doi: 10.3389/fonc.2022.933750. eCollection 2022.
Zinc oxide nanomaterial is a potential material in the field of cancer therapy. In this study, zinc oxide nanospheres (ZnO-NS) were synthesized by Sol-gel method using yeast extract as a non-toxic bio-template and investigated their physicochemical properties through various techniques such as FTIR, XR, DLS, and TEM. Furthermore, free zinc ions released from the zinc oxide nanosphere suspended medium were evaluated by using the ICP-AS technique. Therefore, the cytotoxicity of ZnO nanospheres and released Zn ions on both HuH7 and Vero cells was studied using the MTT assay. The data demonstrated that the effectiveness of ZnO nanospheres on HuH7 was better than free Zn ions. Similarly, ZnO-Ns were significantly more toxic to HuH7 cell lines than Vero cells in a concentration-dependent manner. The cell cycle of ZnO-Ns against Huh7 and Vero cell lines was arrested at G/M. Also, the apoptosis assay using Annexin-V/PI showed that apoptosis of HuH7 and Vero cell lines by ZnO nanospheres was concentration and time-dependent. Caspase 3 assay results showed that the apoptosis mechanism may be intrinsic and extrinsic pathways. The mechanism of apoptosis was determined by applying the RT-PCR technique. The results revealed significantly up-regulated Bax, P53, and Cytochrome C, while the Bcl results displayed significant down-regulation and the western blot data confirmed the RT-PCR data. There is oxidative stress of the ZnO nanospheres and free Zn ions. Results indicated that the ZnO nanospheres and free Zn ions induced oxidative stress through increasing reactive oxygen species (ROS) and lipid peroxidation. The morphology of the HuH7 cell line after exposure to ZnO nanospheres at different time intervals revealed the presence of the chromatin condensation of the nuclear periphery fragmentation. Interestingly, the appearance of canonical ultrastructure features of apoptotic morphology of Huh7, Furthermore, many vacuoles existed in the cytoplasm, the majority of which were lipid droplets, which were like foamy cells. Also, there are vesicles intact with membranes that are recognized as swollen mitochondria.
氧化锌纳米材料是癌症治疗领域的一种潜在材料。在本研究中，以酵母提取物作为无毒生物模板，通过溶胶 - 凝胶法合成了氧化锌纳米球（ZnO - NS），并通过傅里叶变换红外光谱（FTIR）、X射线衍射（XR）、动态光散射（DLS）和透射电子显微镜（TEM）等多种技术研究了其物理化学性质。此外，利用电感耦合等离子体质谱（ICP - AS）技术评估了从氧化锌纳米球悬浮介质中释放的游离锌离子。因此，采用MTT法研究了ZnO纳米球和释放的锌离子对HuH7和Vero细胞的细胞毒性。数据表明，ZnO纳米球对HuH7的有效性优于游离锌离子。同样，ZnO - Ns对HuH7细胞系的毒性明显高于Vero细胞，且呈浓度依赖性。ZnO - Ns对Huh7和Vero细胞系的细胞周期阻滞在G/M期。此外，使用膜联蛋白V/碘化丙啶（Annexin - V/PI）的凋亡检测表明，ZnO纳米球诱导HuH7和Vero细胞系凋亡具有浓度和时间依赖性。半胱天冬酶3检测结果表明，凋亡机制可能涉及内源性和外源性途径。通过逆转录 - 聚合酶链反应（RT - PCR）技术确定凋亡机制。结果显示，促凋亡蛋白Bax、P53和细胞色素C显著上调，而抗凋亡蛋白Bcl显著下调，蛋白质免疫印迹数据证实了RT - PCR数据。ZnO纳米球和游离锌离子存在氧化应激。结果表明，ZnO纳米球和游离锌离子通过增加活性氧（ROS）和脂质过氧化诱导氧化应激。在不同时间间隔暴露于ZnO纳米球后，HuH7细胞系的形态显示核周染色质凝聚和碎片化。有趣的是，Huh7出现了典型的凋亡形态超微结构特征，此外，细胞质中存在许多空泡，其中大部分是脂滴，类似泡沫细胞。同时，还有完整的膜泡，被认为是肿胀的线粒体。
