In vitro anticancer and antibacterial performance of biosynthesized Ag and Ce co-doped ZnO NPs

Abstract

The great potential of zinc oxide nanoparticles (ZnO NPs) for biomedical applications is attributed to their physicochemical properties. In this work, pure and Ag and Ce dual-doped ZnO NPs were synthesized through a facile and green route to examine their cytotoxicity in breast cancer and normal cells. The initial preparation of dual-doped nanoparticles was completed by the usage of taranjabin. The synthesis of Ag and Ce dual-doped ZnO NPs was started with preparing the Ce:Ag ratios of 1:1, 1:2, and 1:4. The cytotoxicity effects of synthesized nanoparticles against breast normal cells (MCF-10A) and breast cancer cells (MDA-MB-231) were examined. The hexagonal structure of synthesized nanoparticles was observed through the results of X-ray diffraction (XRD). Scanning electron microscopy (SEM) images exhibited the spherical shape and smooth surfaces of prepared particles along with the homogeneous distribution of Ag and Ce in ZnO with high-quality lattice fringes without any distortions. According to the cytotoxic results, the effects of Ag/Ce dual-doped ZnO NPs on breast cancer (MDA-MB-231) cells were significantly more than of pure ZnO NPs, while dual-doped and pure nanoparticles remained indifferent towards breast normal (MCF-10A) cells. In addition, we investigated the antimicrobial activity against harmful bacteria.

Graphical Abstract

Introduction

As a novel type of widely used mineral particles [1], metal–organic framework [2] and metallic nanoparticles [3–5] such as zinc oxide were noticed and explored by researchers due to their suitable mechanical [6, 7], physical [8, 9] and chemical properties [10–13] that are combined with a higher adsorption power than other zinc-containing compounds [14, 15]. Zinc oxide is one of the compounds of zinc that was recognized as a safe substance by the US Department of Food and Drug Administration [16]. The properties of nanostructures led to their various applications such as anticancer [17, 18], tissue engineering [19–21], antimicrobial [22, 23], degradation [24], photocatalyst [25–29], antioxidant [30], sensor [31–36], sensing [37–39], agriculture [40–42], absorption [43], purification [44, 45], energy [46–49], anti-inflammatory therapy [50], food analysis [51], and drug carriers [52–54]. Among the notable properties of zinc oxide nanoparticles, one can point out their high chemical stability, low dielectric constant, high catalytic activity, absorption of infrared and ultraviolet light, and most importantly their antibacterial properties [55]. Confirming the therapeutic and toxic effects of these compounds can stand as a significant step throughout the advancements of cancer [56–63] and fungal/bacterial infection disease [64–66] treatments [67–69] such as COVID 19 [70, 71]. The primary prevention of infection [72–74] and cancers’ diseases [75–77], new development in research [78–80] and innovation [81–84], such as nanotechnology [85, 86], materials [87–89] and digital technologies [90], have the need to improve our understanding of diseases [91–93] such as cancer [94–97]. In fact, recent developments [98] in all field of science [99–101] and technology [102–104] have impact on human health [105–108] and life [109–112].

Although the main action mechanism of nanoparticles remains unknown[113–115], yet the results of various studies on in in vivo and in vitro environments [116, 117] were indicative of their ability to produce reactive oxygen species (ROS) [118–120], which consequently points out their potent functionality in intracellular calcium concentration, activation of transcription factors, and alterations in cytokines [121–123]. The various approaches of ROS in damaging cells include DNA damage [124–126], interference with cellular signaling pathways, changes in gene transcription, etc. [127–129].

There are different physical [130], chemical [61, 131], and biological methods [132–137] for synthesizing nanostructures, while the exertion of each technique is dependent on the available conditions and purpose of the synthesis [138]. The most common synthesizing methods for the prepare of zinc oxide nanoparticles are observed in the form of sol–gel [139], microemulsion, mechanical–chemical process, direct solvent evaporation, hydrothermal, and spark deposition, which is selected depending on certain factors such as surface chemistry, size distribution, particle morphology, and particle reaction in solution [140]. Next to the advantages of these procedures, there are disadvantages as well since the involved substances are toxic and their usage in medical research is limited [141, 142]. In addition, some of the applied materials remain insoluble and can cause environmental pollution [143–146]. Therefore, in recent years, the application of biological or green methods was noticeably highlighted in order to overcome the disadvantages [147–151]. Green synthesis is defined as the exertion of biological organisms, such as microorganisms, for completing the synthesizing processes that are composed of different bacteria species, actinomycetes [152], algae, fungi, bacteria [153], and biomass [154] or plant extracts [155–159]. Green synthesizing techniques lack the hazardous aspects of physical and chemical methods, and on the other hand, they were confirmed to be environmentally friendly [160] and cost-effective [161] without requiring the usage of high pressure, high energy, high temperature, and toxic chemicals [162–165]. The application of plant extracts for the synthesis of nanoparticles may be a better option than other biological methods since it is suitable for conducting large-scale synthesis while being more cost-effective, as well as capable of accurately preserving the cellular environment [166–168].

Alhagi persarum is a shrub with thin, branched, and prickly stems with an average height of 50 cm. The leaves of this plant are small, oval, pointed, and simple that grows at intervals from the stems. This plant mainly grows in hot and deserted areas (deserts) of Iran, especially in southern regions. A sugary substance is secreted from the stems of this plant that is known as taranjabin in Iran, which turns into white, yellow, or brownish-yellow droplets upon being exposed to air. The chemical composition of taranjabin includes 47.7% of melezitose, 26.44% of sucrose, 11.64% of fructose reducing sugar, 12.4% of gum, and mucilage and 5.1% of ash. Taranjabin is recognized as a laxative that can relieve rheumatic, chest, cough, fever, and biliary pains, which is used in traditional medicine for the treatment of jaundice in infants, as well as children with rubella and infectious fevers.

In order to discover fast and effective treatment pathways or to produce materials with high therapeutic effects for the treatment of cancer, this study attempted to synthesize Ag and Ce dual-doped ZnO NPs by the usage of taranjabin for the very first time and evaluated the cytotoxic activity of synthesized nanoparticles on human breast cancer (MDA-MB-231) and breast normal (MCF-10A) cells lines. In addition, we investigated the antimicrobial activity against harmful bacteria.

Materials and methods

Synthesis of pure and dual-doped ZnO NPs

The synthesis of Ag and Ce dual-doped ZnO NPs was started with preparing the Ce:Ag ratios of 1:1, 1:2, and 1:4. Then, 0.3 gr of taranjabin was dissolved in 50 mL of distilled water within four Erlenmeyer flasks to arrange one sample of un-doped and three samples of dual-doped nanoparticles, respectively. In the following, subsequent to the addition of zinc nitrate hexahydrate (0.02 M, Zn(NO₃)₂.6H₂O, Merck) to all the four taranjabin solutions, silver nitrate (AgNO₃, Merck) and cerium nitrate hexahydrate (Ce(NO₃)₃.6H₂O, Merck) were appended in accordance with the specified ratios, respectively. Once the solutions were mixed by a heater stirrer at 70 °C for 3 h, they were dried in an oven at 80 °C for 24 h. The resulting raw material was calcined at 600 °C for 2 h. The un-doped and cerium and silver dual-doped ZnO NPs were labeled as ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO, respectively (Fig. 1).

Fig. 1.

Images of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

Characterization

The size, morphology, and other physical–chemical properties of synthesized nanoparticles were examined through the performance of PXRD (Netherlands, PANalyticalX’Pert PRO MPD system, Cu Kα), UV–Vis (Rayleigh: UV-2100, China), Raman spectra that were captured by a Raman Takram P50C0R10 device at the laser wavelength of 532 nm, FESEM (MIRA3 TESCAN, Czech), and UV–visible spectroscopy (UV–Vis, UV-1800, SHIMADZU) analyses.

Cytotoxic

Cells’ culture

In this study, human breast cancer (MDA-MB-231) and breast normal (MCF-10A) cells were used to evaluate the cytotoxicity of synthesized nanoparticles. MCF-10A and MDA-MB-231 cells were obtained from the Pasteur Institute of Iran and thawed in prior to being cultured. The cells were transferred to Falcon tubes and centrifuged at 833 rpm for 9 min. Once the supernatant was removed, a complete culture medium was added to the cells to have the prepared suspensions poured into flasks. High-glucose DMEM culture medium was exerted for the process of cells culturing and the next step required the addition of 10% fetal bovine serum (FBS), 100 μg/mL of streptomycin, and 100 international units/mL of penicillin to each culture medium to prevent the inducement of microbial growth. In order to proliferate and grow the cells, the culture medium was incubated under 5% CO₂ at 37 °C.

MTT assay

Human breast cancer (MDA-MB-231) and breast normal (MCF-10A) cells were cultured in an incubator with a high glucose DMEM that was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (37 ◦C, 5% CO₂) until the cells count of each well of 96-well plate reached 10,000. The culture medium was replaced with 100 μL of the DMEM that contained the formulations at different concentrations (1, 10, 50, 100, and 500 μg/mL) to be seeded for another 24 h. Three duplications were considered for each concentration. In the following, the culture medium was changed after 24 h along with the replacement of fresh high glucose DMEM. Then, 20 μL of 5 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) solution was added to each well and another course of incubation was performed for 4 h. Once 100 µL of DMSO was added to each well of 96-well plate, the resulting mixture was shaken for about 15 min at room temperature to dissolve the formazan. A microplate reader was exerted to measure the optical density (OD) at 570 nm. In addition, the cells viability rate (VR) was calculated according to the following equation:

$$\text{VR}=\text{A}/{\text{A}}_0\times 100\%$$

in which A represents the absorbance of the cells that were treated with formulations and A₀ refers to the absorbance of control group.

Antibacterial assay

The antibacterial test was studied on Pseudomonas aeruginosa using macrodilution method. The P. aeruginosa were cultured on these culture media in contact with nanoparticles. The concentrations of 1–250 mg/mL of nanoparticles were prepared in the Mueller Hinton culture medium. Then, the samples were placed in an incubator at 37 °C for 24 h. Finally, bacterial turbidity in the culture media was observed. The turbidity was a sign of the growth of the microbial strain in that concentration of nanoparticles.

IC₅₀

The conduction of probit test was completed through the exertion of SPSS software for two purposes including the calculation of drug and nanoparticles concentrations that could limit the growth of 50% of cells (IC₅₀) and to measure the restriction percentage of cells growth against concentration.

Results and discussion

XRD analysis

Figure 2 presents the XRD pattern of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles. The observed peaks in pure ZnO nanoparticles and Ag and Ce dual-doped ZnO nanoparticles were indexed to (100), (002), (101), (102), (110), (103), (200), (112), and (201), which is comparable with the hexagonal structure of ZnO (JCPDS-36–1451). In conformity to Fig. 2, increasing the ratio of Ag resulted in the appearance of peaks related to the silver-doped nanoparticles throughout the PXRD pattern. The purity and high crystalline form of synthesized nanoparticles was confirmed by the lack of observing any other additional peaks. The crystalline size of synthesized nanoparticles was calculated through the Debye–Scherer formula as given in the following equation:

$$\text{D}=\text{K}\lambda /\beta \text{cos}\theta$$ 2

Fig. 2.

PXRD pattern of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

where D refers to the crystallite size of nanoparticles, K represents the shape factor, λ is the wavelength of applied radiation, β would be full width at half maxima (FWHM) in radians, and θ stands for the diffraction angle. The average crystallite size of synthesized nanoparticles was estimated by considering the full width at half maxima (FWHM) of XRD peak (101) through the usage of Debye–Scherer formula, which was obtained to be 19.14, 19.73, 22.05, and 22.20 nm for ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles, respectively. The data in Fig. 2 indicate that the doping of Ce and Ag metals to the crystalline network of ZnO nanoparticles caused an increasing in the crystalline size of synthesized doped nanoparticles due to the difference in ionic radius of zinc atom (1.38 Å) when compared to silver (1.26 Å) and cerium (1.037 Å).

FESEM and EDX analyses

Figure 3 presents the FESEM images of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles obtained by the usage of taranjabin, which displays the approximately spherical shape of ZnO particles. The recorded doped nanoparticles throughout the FESEM images were also spherical, while observations indicated the inducement of an increasing in the size of synthesized particles due to the doping of Ag and Ce metals into the structure of ZnO. The mean particle size distribution of synthesized ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles, which were estimated to be 31.59, 31.93, 36.89, and 38.44 nm, exhibits the satisfying growth of particles as a result of increasing the percentage of doped metals. In conformity to the provided EDX profiles of biosynthesized ZnO and Ag4/Ce–ZnO nanoparticles in Fig. 4, the synthesized nanoparticles contained a high-purity content with the composition of Zn and O elements for ZnO and Zn, as well as O, Ag, and Ce elements for Ag4/Ce–ZnO nanoparticles. The table form of elemental composition is inserted in Fig. 4.

Fig. 3.

FESEM images and particle size distribution of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

Fig. 4.

EDX profiles of biosynthesized pure ZnO and Ag4/Ce–ZnO nanoparticles using taranjabin

Raman analysis

Raman spectroscopy is a non-destructive chemical analysis technique for providing detailed information about chemical structure, phase and polymorphy, crystallinity, and molecular interactions, which is based upon the interaction of light with chemical bonds within a material. According to group theory, ZnO nanoparticles contain a hexagonal wurtzite structure with a space group of P63mc. The optical modes of A1 + 2B2 + E1 + 2E2 imply the wurtzite structure of ZnO, which includes A1 + E1 + 2E2 as the active Raman mode, A1 + E1 as the active infrared mode, and 2B1 as the silent Raman mode. The A1 and E1 modes are two polar branches that are divided into longitudinal optical (LO) and transverse optical (TO). The A1, E1, and E2 modes are recognized as the first-order Raman active and based on Raman law, B1 modes are usually inactive throughout the Raman spectrum and are known as the silent modes. The Raman spectra of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles are represented in Fig. 5.

Fig. 5.

Raman spectra of biosynthesized (A) pure ZnO nanoparticles, and (B) ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

The main phonon states of ZnO nanoparticles with a hexagonal structure appeared in the regions of 583, 441, 345, 91 cm⁻¹, which were in correspondence to the A1(LO)–E1(LO), E2H, A1(TO), and E2H modes, respectively. The 2E2L mode was in correlation to the second-order phonon mode that appeared in the region of 132 cm⁻¹. Moreover, the modes of 3E2H-E2L, E1(TO) + E2L, 2(E2H-E2L), and A1(TO) + E1(TO) + E2L were related to the poly-phonon scattering that was detected in the points of 324, 475, 658, and 1105 cm⁻¹, respectively.

As it is displayed in Fig. 5, the doping factor (both Ag and Ce) of ZnO matrix caused significant changes in the polar and non-polar states. The E2H state involves the oxygen motion, while being sensitive to internal stress, and containing the characteristics of hexagonal structure of zinc oxide nanoparticles. Due to the decomposition of impurities and defects, the E2H mode faced a sharp decrease in the peak intensities of doped samples. In addition, this mode was observed to be steadily decreased and expanded as the doping concentrations of silver and cerium were increased. The detected polarity of A1(LO)–E1(LO) at around 583 cm⁻¹ was related to the doping of silver and cerium that can expand a peak and also force its shifting towards lower energies. All the variations and extensions of phonon modes were obtained by scattering contributions outside the center of Brillouin area. The phonon state of A1 (LO)–E1 (LO) is usually attributed to the interfacial defect of zinc and oxygen vacancy throughout the network of ZnO. Due to the combination of Ag and Ce ions with ZnO nanoparticles, the intensity of ZnO Raman peaks can be greatly increased through the doping of silver and cerium. In addition, further results confirmed the crystallization of ZnO nanoparticles with few defects due to the presence of Ag and Ce ions.

UV–Vis analysis

Electron spectroscopy is a technique for investigating the energy distribution of ejected electrons from a material as a result of being irradiated by a source of ionizing irradiation. Figure 6 presents the electronic spectra of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles obtained by the usage of taranjabin. The maximum wavelength of pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles were observed at the regions of 396, 372, 383, and 385 nm, respectively.

Fig. 6.

Electronic graph of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

An increase in the concentration of Ce and Ag throughout the structure of ZnO causes a shifting in the absorption spectra towards higher wavelengths (red shift) due to the induced alteration in the amount of optical bandgap. This red shift represents the increasing crystallization and the effects of quantum confinement. An enlargement in the electron population during the doping of Ce and Ag into ZnO can lead to quantum constraints and finally cause a red shift in optical absorption behavior.

Cytotoxic performance

In this study, we examined the cytotoxicity effects of biosynthesized ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles obtained by the usage of taranjabin against breast normal cells (MCF-10A) and breast cancer cells (MDA-MB-231). For this purpose, the cells were exposed for 24 h at different concentrations (1–500 μg/mL) of un-doped and dual-doped ZnO nanoparticles through the means of MTT assay (Fig. 7). In conformity to Fig. 6, the pure and Ag and Ce dual-doped ZnO nanoparticles did not cause any significant toxicity effects on the normal cell line (MCF-10A), while the doped nanoparticles resulted in almost similar toxicity impacts to that of un-doped nanoparticles. Furthermore, increasing the concentrations of doped and un-doped nanoparticles did not cause any significant toxicity effects. The assessment results of cytotoxic activity of synthesized nanoparticles on breast cancer cell line (MDA-MB-231) are presented in Fig. 7. According to observations, increasing the applied concentration intensified the effects of cytotoxicity, which reached a significant point at the concentration of 500 µg/mL. The cytotoxic effect of doped nanoparticles was more that of un-doped nanoparticles. As, 80% of the cells were killed from being treated with Ag4/Ce–ZnO nanoparticles at the concentration of 500 µg/mL. In addition, IC₅₀ data strongly confirmed the obtained results (Table 1), which less IC₅₀ was attributed to Ag4/Ce–ZnO nanoparticles. Hence, Ag4/Ce–ZnO nanoparticles show the greatest effect of toxicity. Figure 8 depicts the effect of synthesized nanoparticles being treated with breast normal cell and breast cancer cell lines. This figure clearly displays the difference in the cytotoxic activity of synthesized pure and dual-doped ZnO nanoparticles against these two cell lines. These results suggested that the synthesized nanoparticles induced cytotoxicity in cancer cells without affecting the normal cells.

Fig. 7.

The cytotoxic activity of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

Table 1.

IC₅₀ values of biosynthesized pure ZnO, Ag1/Ce–ZnO, Ag2/Ce–ZnO, and Ag4/Ce–ZnO nanoparticles using taranjabin

Cell lines	IC50 values (µg/mL)
	ZnO	Ag1/Ce–ZnO	Ag2/Ce–ZnO	Ag4/Ce–ZnO
MCF-10A	604.2647	799.8132	778.4796	878.4803
MDA-MB-231	447.3	418.113	325.833	220.461

Fig. 8.

Images of breast normal cells (MCF-10A) and breast cancer cells (MDA-MB-231) lines treated with pure ZnO and Ag4/Ce–ZnO nanoparticles after 24 h treatment

The cytotoxic effects of green synthesized doped ZnO NPs on cancer cells were mentioned in the work of many authors. For example, MJ Akhtar et al. studied the oxidative stress-mediated cytotoxicity of Al-doped ZnO nanoparticles against MCF-7 cells. According to their report, Al-doping was able to enhance the cytotoxicity and oxidative stress responses of ZnO nanoparticles against MCF-7 cells. In addition, they obtained an IC₅₀ of 44 μg/mL for un-doped ZnO nanoparticles and 31 μg/ml for the Al-doped ZnO counterparts. It was suggested by their results that Al-doped ZnO nanoparticles can induce apoptosis in MCF-7 cells through the mitochondrial pathway [169]. In another work, G. Vijayakumar et al. investigated the cells viability, ROS generation, and nanoparticle cells penetration rate of PEG encapsulated bare and Mn-doped ZnO nanoparticles against human liver carcinoma Huh7 cell lines. Based on their findings, un-doped the Mn-doped ZnO nanoparticles exhibited a higher cells annihilation effect, which may be due to the combined effects of Zn²⁺ ion release and intracellular ROS generation; therefore, the inducement of apoptosis can be expected due to oxidative stress and ROS generation [170]. Considering these facts, doped metal oxide nanoparticles can stand as an attractive research topic for biomedical applications. Nano-sized materials have enabled many developments in biomedicine and other biological applications such as drug delivery, anticancer activity, gene delivery, fluorescent biological labels, protein detection, MRI contrast enhancement, probing of DNA, tissue engineering, phagokinetic studies, hyperthermia, and filtration of biological based molecular cell.

The antibacterial test of doped and non-doped nanoparticles was on P. aeruginosa and E.coli. The IC₅₀ was at 50 μg/mL.

Conclusion

Un-doped and Ag and Ce dual-doped ZnO NPs were synthesized through a facile green method by exerting the extract of taranjabin. The obtained PXRD spectra displayed the hexagonal phase of un-doped and dual-doped ZnO NPs. SEM mapping demonstrated the homogeneous distribution of Ag and Ce in ZnO with high-quality lattice fringes while lacking any distortions. According to cytotoxicity results, the un-doped ZnO NPs displayed a similar toxicity effect on breast cancer cells (MDA-MB-231) to that of dual-doped ZnO NPs. Considering the comparable toxicity effect of doped nanoparticles with the un-doped nanoparticles, it can be stated that the simultaneous doping of cerium and silver did cause significant alterations in the cytotoxic properties of zinc oxide nanoparticles. However, this discovery requires further investigation since it may affect certain physical and biological properties such as luminescence, UV absorption, or antibacterial features of zinc oxide nanoparticles. Therefore, this attempt can stand as a useful approach due to the cosmetic and even industrial applications of zinc oxide nanoparticles.

Declarations

Conflict of interest

The authors confirm that the content of this article involves no competing interests.