Introduction

A type of brain tumor known as glioma arises from glial cells, which supports neurons and include ependymal, oligodendrocyte, and astrocyte cells. Gliomas constitute approximately 30% of all brain tumors and 80% of malignant brain tumors. Grade IV astrocytoma, also known as glioblastoma (GBM), is the most common type of glioma, representing over 60% of all brain tumors1. The incidence rate of GBM- the most prevalent and aggressive malignant tumor of the central nervous system (CNS)- has increased over time, ranging from 0.59 to 5 cases per 100,000 individuals. As individuals age, the incidence of GBM rises gradually. Typically, the probable survival period is 12 to 18 months with only 5% of diagnosed individuals living longer than five years. Glioblastoma is usually diagnosed at an advanced stage, limiting effectiveness of surgical procedures1,2,3,4. Patients with GBM have a poor prognosis and a low survival rate, despite advances in oncology. The median survival for GBM patients has remained relatively unchanged over the past 25 years. population studies, have shown that 60–70% of patients have a median survival of only 12 months with multimodal therapy options, and fewer than 2 years in clinical trials1. Thus, GBM remains a fatal illness with no current cure2,3. Surgery, chemotherapy (temozolomide) and radiotherapy are commonly used treatments for GBM, but resistance is common5. Radiotherapy alone is not very effective and poses a high risk of damaging healthy brain tissue. Complete tumor removal is often impossible, leading to a high chance of recurrence. Scientists and medical professionals are continuously researching the molecular processes and heterogeneity of GBM to improve treatment outcomes1,2.

In recent years, nanotechnology has emerged as an exciting area of research, particularly in the field of nanomedicine. Nanoparticles (NPs) show promise in enhancing cancer therapy, diagnosis, and prevention. Due to their ability to target cancer cells, Researchers are exploring the use of NPs in developing new anti-cancer medications to combat this devasting disease. The unique properties of NPs such as their high surface to volume ratio and small size offer potential for improving cancer treatment outcomes6,7. Among several synthesized NPs, metal oxide NPs possess important advantages, drawing interest from researchers for their potential use in nanomedicine. These advantages include the ability to penetrate cells, porosity, size, engineering potential, high stability, and ease of synthesis. Because of these advantages, MONPs have outperformed their bulk counterparts in terms of limitations. One of the most significant MONPs is zinc oxide nanoparticle, which is biocompatible and non-toxic, and has been extensively used in a variety of commercial products. Several studies have demonstrated that ZnO NPs exhibit specific cytotoxicity against cancer cells while leaving normal cells unharmed8,9. as a result of this significant behavior, these NPs have been FDA approved as an anti-tumor treatment10. Apoptosis is one of the major pathways participating in cell death caused by ZnO NPs9.

ZnO NPs have been shown in numerous studies to exhibit significant cytotoxic effects against various cancers. According to a study by R.Yanag et al., ZnO NPs can considerably eliminate cancer cells in the Huh-7 cell line by up-regulating P53 and inducing apoptosis. Thus, they may be useful as a treatment for liver cancer11. According to a 2014 study, ZnO NPs have a high cytotoxic effect on U373 GBM cell line through apoptosis12.

Traditional methods have been employed to synthesize NPs in the last years. In these methods not only the usage of carcinogenic chemicals was a concern but also because of the high energy input, they are considered as costly NP synthesis approaches. As of this reason, researchers have been seeking novel biocompatible methods to synthesize NPs that have the least harmful effects on human and nature. Green synthesis is a highly efficient method, maybe even more efficient than traditional methods. This approach uses natural sources and molecules, providing a biocompatible, cost-effective, and safe way to synthesize NPs13.

Doping an element into a nanoparticle causes significant changes in its size and morphology14. The doping of metals into NPs has attracted attentions due to the improvement of characteristics and functions of these NPs. Studies have shown that ZnO NPs doped with different metals exhibit higher anti-bacterial activity compared to those that are not doped15.

In this study, our aim is to synthesize sodium doped zinc oxide NPs using zingerone in an environmentally friendly manner. We will characterize their novel properties and evaluate their cytotoxic effects on U87 GBM cell line and normal HEK cell line. Additionally, we will investigate the occurrence of apoptosis and cell cycle regulation through gene expression study of TP53 and related genes to apoptosis and cell cycle regulation pathways.

Materials and methods

Nanoparticle synthesis

To synthesize sodium-doped zinc oxide NPs, 2 g of zingerone (SIGMA) were dissolved in 100 mL of ethanol. Next, 6 g of zinc acetate were dissolved in the ethanolic zingerone solution using a magnetic stirrer followed by the addition of 0.3 g of NaNO3. The solution was then stirred at 70 °C for 3 h on a heater stirrer. The resulting mixture was dried in an oven at 70 °C. Finally, it was calcined at 700 °C for 3 h and turned into powder using a mortar and pestle.

Nanoparticle characterization

The physical properties and particle size of the green synthesized NPs were assessed using DLS (dynamic light scattering) and XRD (X-ray diffraction). Fourier-Transform Infrared Spectroscopy (FTIR), was conducted to confirm the presence of functional groups. Field Emission Scanning Electron Microscopy (FESEM) was used to analyze the shape and surface morphology of the sodium-doped zinc oxide NPs.

Cell lines and cell culture

The U87 GBM cancer cell line and HEK as the control cell line were purchased from Pasteur institute and used in this research. The cells were cultured in T-25 flasks using DMEM/F12 medium (Dulbecco’s Modified Eagle’s Medium) containing 10% FBS (fetal bovine serum) and 1% antibiotic (penicillin/ streptomycin). The cells maintained in a humidified incubator set at a suitable temperature (37 °C), with 5% CO2.

MTT assay

MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was executed in order to evaluate the cytotoxic effect of Na-doped ZnO NPs on the viability of U87 and HEK cell lines. To this purpose, the cells were seeded onto 96-well plates. After 24 h, cells were treated with a serial concentration (0, 10, 20, 50, 100, 200, 400, 800, and 1000 µg/ml) of Na-doped ZnO NPs. The cells were incubated for 48 h, and then MTT solution (5 mg/mL) was added to each well and incubated in the dark for 3 h before gently replacing each well’s contents with 100 µl of DMSO. The formazan crystals of MTT reduction were dissolved in DMSO and their absorbance was measured using a microtiter plate ELISA reader (BioTek). Polynomial regression was used to calculate the IC50 of Na-doped ZnO NPs against U87 and HEK cells.

Real time PCR

mRNA expression pattern of regulatory genes such as TP53, P21, PTEN, BAX, and Bcl2 was used for apoptosis and cell cycle pathway analysis. cells were treated with the IC50 of Na-doped ZnO NPs for 24 and 48 h, and then RNA was extracted with Trizol, according to the manufacturer’s protocol (Kiagene- Cat. No: FPLR034.0025). Complementary DNA was synthesized using oligo dT primers (Kiagene- Cat. No: FPLF012.0025). Quantity of genes expression was determined by quantitative Real-time PCR using Syber Green-I (Kiagene- Cat. No: FPLF009.1000) by means of the Rotor-GeneTM 6000 machinery (Corbett research, Australia). The Beta-Actin mRNA was selected as the reference gene. primer sequences of genes used in real-time PCR were shown in Table 1. Relative quantitative calculation of gene expressions was carried out using Pfaffl equation16 that is mentioned below, in which R is relative expression and E represents PCR efficiency. Real-time PCR was performed twice, each time in triplicate.

$$\:R={E}^{\varDelta\:\varDelta\:ct}\to\:R=\frac{{E}_{Target}^{\varDelta\:ct}}{{E}_{Refrence}^{\varDelta\:ct}}=\frac{{E}_{Target}^{{ct}_{Control}-{ct}_{Sample}}}{{E}_{Refrence}^{{ct}_{Control}-{ct}_{Sample}}}$$
Table 1 Primer sequences of target genes whose relative expression levels were measured using real-time PCR.
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Apoptosis assay by flow cytometry

Apoptosis and necrosis were determined after 24 and 48 h of treatment with the IC50 of sodium-doped zinc oxide NPs following the manufacturer’s instructions. Cells were trypsinzed, then centrifuged (5 min, 1200 RPM) for collection. The cells were washed with cold PBS three times. Binding buffer X (MabTag- Cat. No: AnxBuff) was added to each tube to resuspend cells. 100 µl of each cell suspension was added separately into flow cytometry tubes. 3 µl of FITC Annexin V (MabTag- Cat. No: AnxF100) solution was added to each tube and kept in the dark for 20 min. 3 min before the test, 1.5 µl of PI (MabTag- Cat. No: AnxF100) solution was added to each tube. Samples were then analyzed by flow cytometry.

Statistical analysis

Experiments were performed in duplicate, and the results were presented as mean ± SEM. Normality of the data was analyzed using Shapiro-Wilk test. The results were statistically analyzed by one-way ANOVA test. A p-value of 0.05 or lower is generally considered statistically significant. The statistical analysis of the data was performed using Prism software.

Results

Characterization of Na-doped ZnO NPs

FESEM

FESEM was conducted to evaluate the shape, morphology, and size of green-synthesized Na-doped ZnO NPs. Based on the data presented in Fig. 1, it can be concluded that the NPs display an aggregative nature and have a spherical shape. The average size of the NPs is estimated to be 55.39 nm. The diameter distribution of the NPs is shown in Fig. 2.

Fig. 1
figure 1

FESEM images of green synthesized Na-doped ZnO NPs at various magnifications indicate that Na-doped ZnO NPs display an aggregative nature and have a spherical shape.

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Fig. 2
figure 2

Diameter distribution of green synthesized Na-doped ZnO NPs with an average size of 55.39 nm.

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XRD

The XRD spectrum of green synthesized Na-doped ZnO NPs is presented in Fig. 3. The various diffraction peaks at (2θ) were 31.89°, 34.45°, 36.31°, 47.59°, 56.62°, 62.88°, 66.47°, 67.94°, 69.06°, 72.58°, and 76.95°, corresponding to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) planes, which confirm the presence of hexagonal wurtzite structure of ZnO with the calculated lattice constant values of a = b = 3.25 and c = 5.196 based on the standard data (JCPDS # 36-1451). The outcomes demonstrated the outstanding crystallinity of the NPs. The crystallite size of the NPs was calculated using Debys-Scherer’s equation and W-H plot.

Debys-Scherer’s equation:

$$\:D=\frac{K.\lambda\:}{\beta\:.Cos\theta\:}$$

where D stands for the crystallite size (nm), K for Scherer’s constant (0.9), β for the FWHM of the diffraction peak, λ for the X-ray source’s wavelength (0.14606, nm), and θ for Bragg’s angle. Using Debys-Scherer’s equation, the crystallite size of the sodium-doped zinc oxide NPs was determined to be 17.56 nm. The Williamson and Hall plot is presented in Fig. 4. The average crystallite size (D) and the micro strain (ε) of the Na-doped ZnO NPs were calculated from the Y-intercept and the slope of the W-H linear plot, respectively. Based on the W-H plot, the average crystallite size of the NPs is 13.69 nm, and the micro strain (ε) value is -0.00116.

Fig. 3
figure 3

XRD spectrum of Na-doped ZnO NPs which confirm the presence of hexagonal wurtzite structure of ZnO.

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Fig. 4
figure 4

W-H plot of Na-doped ZnO NPs. The average crystallite size of Na-doped ZnO NPs calculated based on W-H plot is 13.69 nm.

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DLS

It is well known that the most popular method for analyzing the distribution of NPs is dynamic light scattering (DLS). DLS analysis indicated an average hydrodynamic size of 435.2 nm and a low polydispersity index of 0.08921 for green synthesized Na-doped ZnO NPs.

Zeta potential

Zeta potential analysis was conducted to evaluate the surface charge and physical stability of the NPs in solution. A minimum zeta potential value of ± 30 mV is necessary for a stable nanosuspension17. The results demonstrated that the green synthesized NPs possess a negative surface charge with a zeta potential value of -17.88 mV.

FTIR

FTIR spectrum of the green synthesized sodium-doped zinc oxide NPs is shown in Fig. 5 which shows several peaks in the so-called functional group region such as 3430 cm− 1, 2928 cm− 1, 1728 cm− 1, and a certain peak at 437 cm− 1 in the fingerprint region.

Fig. 5
figure 5

FTIR spectrum of Na-doped ZnO NPs.

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Cytotoxicity of sodium-doped zinc oxide NPs by MTT assay

The effects of the sodium-doped zinc oxide NPs on U87 and HEK cells viability were examined after 48 h of exposure, by using the MTT assay. The MTT assay results showed that cell viability and growth were affected by different concentrations of Na-doped ZnO NPs (Fig. 6). Exposure to sodium-doped zinc oxide NPs led to a decrease in the survival of U87 cells in a dose-dependent manner, as detailed in Fig. 6. The results show that sodium-doped zinc oxide NPs at 38.7 µg/mL decreased the viability of U87 cells to 50% of the initial level, and this was chosen as the IC50. Longer exposures resulted in additional toxicity to the cells while the IC50 for HEK cell line was 86.5 µg/mL (P value < 0.05). The result suggested that the green-synthesized sodium-doped zinc oxide NPs possess great selective cytotoxicity against U87 cancer cells.

Fig. 6
figure 6

Cell viability of HEK and U87 cell lines treated with green synthesized Na-doped ZnO NPs and original pictures of HEK(A) and U87(B) cells treated with different concentrations of Na-Doped ZnO NPs (a control; b 20 µg/ml; c 80 µg/ml; d 600 µg/ml). (Magnification: 40x)

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Altered expression levels of Tp53, P21, Bax, Bcl2, and PTEN in U87 glioblastoma cell line

The mRNA expression levels of apoptosis-related and cell cycle-related genes which are TP53, P21, PTEN, Bax, and Bcl2 were investigated in 24 h and 48 h treated U87 cells. According to our data, in 48 h-treated cells, the expression levels of TP53, BAX, Bcl2, P21, and PTEN were higher in treated cells in comparison to untreated cells (p < .01). On the other side, in 24 h-treated cells, BAX and PTEN expression levels did not show a meaningful difference compared to the control cells while a meaningful decrease (0.72 ± 0.04-fold) in Bcl2 expression levels was observed (Fig. 7). The relative expression levels of target genes are presented in Table 2. The Bax/Bcl2 ratio in U87 cells treated for 24 h with Na-doped ZnO NPs was 1.375, while in cells treated for 48 h, it was 1.622. This ratio at both time points favors apoptosis and indicates a higher induction of apoptosis at 48 h compared to 24 h. These results demonstrate that the sodium-doped zinc oxide NPs that we have synthesized, strongly induce apoptosis through the intrinsic apoptosis pathway at the mRNA level.

Table 2 The relative expression levels of target genes in U87 cells treated with Na-doped ZnO NPs at 24 and 48 h.
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Fig. 7
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Relative mRNA expression levels of TP53, P21, PTEN, BAX, and Bcl2.

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Na-doped ZnO NPs induced apoptosis in U87 cells

Flow cytometry was used to evaluate the induction of apoptosis in U87 cells treated with the IC50 of Na-doped ZnO NPs for 24 and 48 h. The results of this assay are illustrated in Table 3; Fig. 8. The percentage of cells in early apoptotic stage significantly increased at both times, 24 h (22.93 ± 1.93%) and 48 h (43.6 ± 0.53%) compared to the negative control (13.3 ± 0.5%). Additionally, the percentage of late apoptotic cells increased in a time-dependent manner, 24 h (4.35 ± 0.96%) and 48 h (7.76 ± 0.23%) in comparison to the negative control (1.86 ± 0.21%).

Table 3 The percentage of different cellular conditions in U87 cells treated with Na-doped ZnO NPs at 24 and 48 h, measured by apoptosis assay.
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Fig. 8
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Apoptosis assay results in U87 cells treated with Na-doped ZnO NPs at 24 and 48 h. (a: control cells); (b: 24-h treated cells); (c: 48-h treated cells).

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Discussion

In this study, Na-doped ZnO NPs were successfully synthesized using zingerone as the reductive agent. Several characteristics of these NPs such as shape and morphology, crystallinity, size, surface charge, and functional groups were assessed by FESEM, XRD, DLS, Zeta potential, and FTIR. FESEM was conducted to assess size, shape, and morphology of the green synthesized NPs and the results indicated that the Na-doped ZnO NPs have an aggregative nature which has been commonly demonstrated in several studies18,19,20. Nanoparticles possess a vaster surface area that leads NPs to stick together more firmly which results in aggregation19. Based on results of FESEM, these green-synthesized Na-doped ZnO NPs were spherical in shape which according to several studies is a common shape among ZnO NPs18,19,21,22.

The XRD results confirm the presence of ZnO and hexagonal wurtzite structure of nano-scale Na-doped ZnO NPs based on JCPDS card (No. 36-1451)18,19,21. Several different studies have reported different crystallite sizes of ZnO NPs based on XRD results. A study in 2022 showed that the crystallite size of ZnO NPs is 11 nm17 which is approximate to the crystallite size of the NPs in this study. Another study in 2022 has reported the crystallite size of the zinc oxide NPs to be 30–40 nm23. Ragapriya et al. synthesized a ZnO nanoparticle which its size was 205 nm24.

DLS analysis demonstrated a proper uniformity of Na-doped ZnO NPs19. Several former studies showed that DLS average size of ZnO NPs was higher than that of XRD and electron microscopy. This occurs because of the hydrodynamic character of NPs18. For instance, a 2022 study showed that ZnO NPs crystallite size was 52 nm while their average hydrodynamic size was 154 nm18. Tomas et al. reported that ZnO NPs had a crystallite size of 89.97 nm and a hydrodynamic size of 691.1 nm which are significantly different17.

The results of zeta potential indicated a highly stable solution of NPs17.

The Fig. 5 shows the FTIR spectrum of Na-doped ZnO NPs in which the broad peak at 3430 cm− 1 indicates the presence of alcohol and phenolic OH functional groups of which can be implied that the NPs of interest are caped with the alcohol in the synthesis mixture. The peaks at 2960 cm− 1, 2928 cm− 1, and 2860 cm− 1 confirm the presence of C-H bond of alkyl groups and the peak at 1728 cm− 1 is related to C = O. Peaks between 400 and 600 cm− 1 are related to stretching frequency of Zn-O bond so the certain peak at 437 cm− 1 could confirm the presence of Na-doped ZnO NPs18,25,26.

Cytotoxic effects of Na-doped ZnO NPs on U87 and HEK cells after 48 h of treatment were investigated using MTT assay and the results showed that these NPs significantly eliminate U87 cancerous cells (IC50 = 38.7 µg/ml) while having a rather mild cytotoxic effect on HEK cells (IC50 = 86.5 µg/ml). Wahab R et al. reported that ZnO NPs had an IC50 of 61 ± 1.4 µg/mL and 125 µg/mL for U87 and HEK cells, respectively26. A 2023 study by Hamidian et al. showed that the IC50 of ZnO NPs against U87 cells was 291.7 µg/mL27. Another report in 2013, demonstrated an IC50 of 52.9 µg/mL for ZnO NPs against HEK cells25. Comparing the results of the current study to the mentioned literature, it can be deducted that Na-doped ZnO NPs are more efficient at eliminating U87 GBM cells while sometimes being less harmful to normal human embryonic kidney (HEK) cells than ZnO NPs.

The relative expression levels of TP53, P21, PTEN, Bcl2, and BAX in U87 cells that were treated with the Na-doped ZnO NPs for 24 and 48 h were measured by real-time PCR (Fig. 7). Numerous studies reported the regulation of the genes mentioned above in various cell lines due to the treatment with ZnO NPs (Table 4). It was observed in the 24-hour-treated cells that TP53 and P21 were extremely up-regulated and Bcl2 was down-regulated, and this indicates the initiation of the induction of apoptosis and cell cycle regulation28,29,30. The tumor suppressor gene PTEN was upregulated in 48-hour-treated cells, which leads to growth inhibition and apoptosis31. A rather quick up-regulation of TP53 and P21 in comparison to other genes is because both of them are among the main and primary genes to take part in the initiation of apoptosis and cell cycle regulation. Additionally, a higher up-regulation of these genes at 24 h compared to 48 h could have occurred because the induction of cell cycle arrest provides enough time for U87 cells to repair the damage caused by the treatment with Na-doped ZnO NPs28,29,32. Bax was up-regulated in 48-hour-treated cells, which indicates the advancement of apoptosis30. The Bax/Bcl2 ratio indicates apoptosis induction at both time points, with a higher occurrence at 48 h compared to 24 h. This is clearly observed in the flow cytometry results, and these two findings mutually support each other.

Table 4 Various genes regulated by ZnO NPs.
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Conclusions

In this study, Na-doped ZnO NPs were synthesized using a green approach with zingerone as the reductive agent. Multiple characterization analyses confirmed the successful synthesis of the NPs of interest. Based on the results of this study and their comparison with other related studies, it can be concluded that the novel Na-doped ZnO NPs exhibit more targeted cytotoxic effects against U87 cancer cells compared to normal cells. Furthermore, the findings from real-time PCR and flow cytometry suggest that these NPs induce apoptosis, leading to the elimination of U87 cancer cells. Additionally, considering the observed changes in the expression of P21 and PTEN genes, it is predicted that these nanoparticles may also play a role in cell cycle arrest. However, to fully elucidate the precise mechanism of action of Na-doped ZnO NPs, further investigations focusing on key cellular signaling pathways are necessary. Since nanoparticles with a size of less than 100 nm, or more specifically between 20 and 50 nm, are generally considered suitable for crossing the blood-brain barrier (BBB), the Na-doped ZnO NPs synthesized in this study, with an average size of 55.39 nm, could serve as promising candidates for BBB penetration. However, their negative surface charge may present a significant challenge to their transport across the BBB. Therefore, to enhance their potential for glioblastoma treatment and other brain tumors, it is recommended that improved methods be used for the synthesis of nanoparticles that produce NPs with a size range of 20–50 nm and a neutral or slightly positive surface charge to enhance their potential for glioblastoma treatment and other brain tumors. In addition to these recommendations, to establish a comprehensive understanding of the cytotoxic effects of Na-doped ZnO NPs against brain tumors, further investigations should be conducted on other brain tumor cell lines, such as U373, 131N1, T98G, A171 and more. Moreover, in vivo studies using mouse models are also recommended.

In conclusion, it can be asserted that the unique physicochemical properties of NPs turn them into fascinating agents to treat GBM. It is possible for NPs to have the potential to overcome the barriers in the path of treating GBM, such as the blood-brain barrier (BBB). Hence, the necessity of exploring the vast, yet unknown field of nanoparticle potentials cannot be overlooked.