Characterizations of the Synthesized ZnO QD NPs in Nanofluid Product
Due to the significant benefits of nanotechnology in cancer research, we aimed to develop a new form of ZnO nanoparticles (NPs) to study their impact on leukemia, specifically acute promyelocytic leukemia (APL). We synthesized ZnO Q-dots in our lab using simple chemical methods like sol-gel and hydrothermal processes. Additionally, we employed a food-grade polymer called polyvinylpyrrolidone (PVP) to stabilize the formulation of the nanoparticles (NPs) and enhance their therapeutic properties.
Figure 1a illustrates Fourier transform infrared (FTIR) spectroscopy and the optical features of the nanoproduct (NFP NPs), revealing the functional groups present. The peak at 442 cm-1 indicates the hexagonal stretching vibration of Zn–O, while the peak at 1629 cm-1 corresponds to C = O stretching vibrations. The broad peak around 3458 cm-1 signifies –OH stretching and carboxylic acid groups due to water adsorption on the surface of ZnO Q-dots and nanopolymer. Vibrations at 2912 and 2856 cm-1 indicate –CH and –CH2 stretching bands, and the wide band at 651–576 cm-1 is linked to ZnHO defect surface structure and oxygen vacancies of ZnO Q-dots (18).
The UV–Visible spectroscopy results (Fig. 1b) demonstrate absorption peaks at 239 and 282 nm, possibly attributable to the high band gap energy, quantized transition states, and blue emission. This suggests the agent's ability to generate oxygen radicals (ROS) and excellent diffusion in biological applications. Overall, the findings confirm that the ZnO Q-dots have a Wurtzite structure with a hexagonal phase crystallized form, as indicated by the XRD pattern with standard card number JCPDS No. 36-1451, possessing lattice constants of a = b = 3.249 Å and c = 5.206 Å and a space group p63mc (19, 20).
Further analysis using a field emission scanning electron microscope (FESEM) (Sigma VP model, Zeiss, Germany) revealed that the synthesized ZnO Q-dots and nanopolymers exhibit a uniform spherical shape at different magnification levels. Additionally, the small size of ZnO Q-dots was confirmed by transmission electron microscopy (TEM).
Synergistic activity of ZnO NF in combination with ATO in NB4 cells
Inhibition of viability > 50% of controls was indicative of additive or enhanced inhibition of growth since ZnO NF and ATO were augmented at the concentration known to result in 50% inhibition when tested individually. We found that combinational treatment of ZnO NF (5µg/ml) with ATO (1 µM) resulted in 83% inhibition of cell viability compared with control (P ≤ 0.05). Analysis of fraction-affect (FA) versus combination index (CI) and isobologram was determined to assess synergy and antagonism. Our results demonstrated a synergistic anti-proliferative effect when NB4 cells were concurrently treated with ATO and ZnO NF.
The stimulatory effect of ZnO NF on ATO-induced cytotoxicity in NB4 cells was coupled with the production of ROS.
Most mechanisms underlying chemotherapeutic agents involve ROS production, therefore endorsing oxidative impairment (19). Besides, recently, we perceived that both ZnO NF and ATO could induce ROS in the NB4 cell line (20, 21). Therefore, we were interested in investigating whether combining ZnO NF and ATO dramatically affected ROS-induced cell toxicity. To study the oxidative influence of ZnO NF and ATO on cellular oxidation–reduction (redox) state, we measured cellular ROS levels after exposing NB4 cells to ZnO NF, ATO, or a combination of both for 24 hours. We found that ZnO NF alone or ATO alone resulted in an immediate elevation of cellular ROS levels, while combination treatment exhibited a more pronounced effect.
Given that the systemic response to caloric deprivation in cancers is a metabolic switch on the road to the oxidation of lipids released from adipose tissue stores, SIRT1 seems to induce, at least in part, metabolic pathways triggered by low energetic levels. (22). Finding that the expression levels of SIRT1 and other metabolically relevant SIRT1 targets such as FoxO3a were also increased in response to combination treatment as compared with either agent alone (Fig. 2) elucidate the capability of this agent in the induction of oxidative stress in NB4 leukemic cells.
ZnO NF augmented ATO-induced apoptosis through the alteration of apoptosis-related genes.
Notably, high levels of ROS can cause damage to deoxyribonucleic acid (DNA) in the nucleus and mitochondria (23). p53 plays a dual role in stress response by regulating several genes into cell-cycle arrest (24, 25). It activates apoptosis in the case of DNA damage (5). Given the roles of several pro- and anti-apoptotic genes in cell death and survival and to understand the molecular events contributing to the apoptosis, we investigated the effect of the p53, Bax, Bid, PUMA, Bcl2, MCL1, and Survivin genes expression in response to ZnO NF, ATO, or a combination of both. The combination treatment showed significant up-regulation of all tested apoptotic genes, such as p53, Bax, Bid, and PUMA, and down-regulation of anti-apoptotic genes, including Bcl2, MCL1, and Survivin. We found that treatment with both agents in a combined-modality increased the percentage of annexin-V/PI double-positive NB4 cells compared to either agent alone.
The combination of ZnO NF and ATO promoted autophagy of NB4 cells through genes encoding regulators of autophagy.
Induction of autophagy by accumulation of ROS has been well-established in previous studies (26). To investigate whether the synergistic effect of cell death caused by combination treatment induces autophagic cell death, we examined the expression of various autophagy genes such as Pin1, ATG-7, ATG-10, and Beclin-1. The results of qRT-PCR analysis uncovered that the combination-treatment group robustly boosted the expression levels of the designated genes compared to its monotherapy controls. On the other hand, some evidence has proposed that the IRE1/JNK branch of ER stress response contributes to autophagic cell death in cancers (27). Hence, our finding was further strengthened by the results of the expression levels of the IRE1 and JNK. In agreement, the qRT-PCR analysis revealed that the indicated genes' expression levels were changed more significantly when NB4 cells were treated with ZnO NF and ATO in a combined model, as seen in Fig. 2.
The stimulatory effect of ZnO NF on ATO-induced cytotoxicity in NB4 cells was coupled with G2/M Arrest.
In preliminary screening, growth inhibitory effects of simultaneous ZnO NF and ATO treatment were recorded. To determine the possible anti-proliferative mechanism, dose 5µg/ml of ZnO NF and dose 1µM of ATO were selected because they had noteworthy inhibitory effects on the proliferation potential and cytotoxic effects on NB4 cells. Treatment of NB4 cells with ZnO NF and ATO in a combined modality for 24 h resulted in a significantly higher number of cells in the G2/M phase compared to controls (Fig. 3). The effect of drugs combination on G2/M phase arrest in NB4 cells was mainly at the expense of the S and G0/G1 phases. To examine the molecular mechanism(s) underlying variations in cell cycle patterns, we examined the effect of drug combinations on the expression of cyclin-dependent kinase inhibitors p21 and p27. As shown in Fig. 3, the levels of p27 and p21 were enhanced upon treatment with indicated drugs. To strengthen our results, we also measured the replication rate of DNA in NB4 using colorimetric BrdU cell proliferation assay. The resulting data revealed that 24 h treatment with concomitant treatment forced a considerable decrease in the DNA synthesis rate, which is in agreement with the decreased proportion of the cells in the S phase of the cell cycle (Figs. 4 and 5)