Green synthesized Zingiber officinale-ZnO nanoparticles: anticancer efficacy against 3D breast cancer model

Abstract

Aim: ZnO NPs were prepared via green synthesis utilizing Zingiber Officinale.

Methodology: Physical characterization and biological activity were performed against 2D, and 3D spheroids MCF-7 cell lines.

Results: The NPs exhibited 188.9, 175.7 and 171.2 nm size with charge of -8.2, -11.7 and -9.7 mV for the 2%, 3% and 4% formulations. XRD confirmed a wurtzite hexagonal phase. FTIR spectra showed Zn-O stretching vibrations. The 2%, 3% and 4% formulations presented IC₅₀ values of 14.7, 26.2 and 47 μg/ml, respectively, with complete destruction of MCF-7 spheroids. Elevated TNF-α levels suggested an inflammatory-mediated mechanism of action.

Conclusion: 2% Zingiber officinale-derived ZnO NPs showed antitumor potential against deserving further mechanistic and in vivo explorations.

Plain Language Summary

We created tiny particles from ginger extract to test their ability to fight breast cancer. These particles were carefully studied to understand their size, stability and structure. Our tests showed that these ginger-based particles effectively killed cancer cells in a more realistic 3D model, suggesting they might be a powerful new treatment for breast cancer. Further studies are needed to explore their potential use in real-life treatments.

Plain language summary

Article highlights.

Materials & methods

• In this study we synthesized ZnO NPs via green synthesis utilizing Zingiber Officinale.

• ZnO NPs physical characterization and biological activity evaluation against 2D and 3D spheroids MCF-7 cell lines were performed.

Results & discussion

• ZnO NPs showed an average sizes of 194.6, 188.9 and 171.2 nm for the 2%, 3% and 4% formulations, respectively.

• The XRD, EDX and SEM analyses affirmed the successful synthesis of nanoparticles with good morphology and size.

• The 2%, 3% and 4% formulations presented IC₅₀ values of 14.7, 26.2 and 47 μg/ml, respectively with complete destruction of MCF-7 spheroids.

• Elevated levels of TNF-α suggesting an inflammatory-mediated mechanism of action.

Conclusion

• 2% Zingiber officinale-derived ZnO NPs showed an outstanding antitumor potential against MCF-7 cells deserving further mechanistic and in vivo explorations.

1. Introduction

Breast cancer remains a leading cause of mortality among women worldwide, necessitating relentless efforts in the exploration of innovative therapeutic strategies [1–3]. Traditional chemotherapeutic agents, while potent, are often beset with systemic toxicity and adverse effects that limit their efficacy and patient quality of life [4–6]. The continuous need for targeted and less toxic treatments has led to the emergence of nanotechnology as a revolutionary approach in cancer therapy [7–10].

The nanosized particles have novel physicochemical properties compared to their corresponding bulk materials. There are many applications for nanoparticles in the medical field like biosensors, drug delivery systems, imaging, cancer treatment and diagnostic tools [11–13]. The ratio of surface area to volume in nanoparticles is high, which gives them unique and improved properties and expands their potential biomedical applications. In biomedical applications three main groups of nanoparticles are used that are classified based on their chemical structure [14,15]. These nanoparticles include organic nanoparticles such as polymers and liposomes, inorganic nanoparticles such as metals, metal oxides, quantum dots, ceramics and carbon-based nanoparticles [16–18]. The synthesis of metallic nanoparticles can be achieved through various methods such as chemical reduction, green synthesis, sol-gel, pyrolysis, nanoemulsion method, electrochemical synthesis, hydrothermal method, laser ablation, chemical vapor deposition, template-assisted synthesis, ball milling and photochemical synthesis [2,12,19,20]. Among the novel nanomaterials, zinc oxide nanoparticles (ZnO NPs) have garnered substantial interest due to their unique physicochemical properties, biocompatibility and therapeutic potential [21,22]. The potential of ZnO NPs as a treatment for cancer has captured the attention of the scientific community, with numerous studies documenting their effectiveness in combating cancerous cells [23]. Zinc oxide (ZnO) nanoparticles have several favorable properties and specific advantages over other types of nanoparticles in terms of efficacy, selectivity, biocompatibility and other relevant factors [24]. In term of anticancer efficacy ZnO nanoparticles exhibit photocatalytic activity, generating reactive oxygen species (ROS) under UV irradiation. This property can be utilized for targeted cancer cell destruction through ROS-induced apoptosis. ZnO nanoparticles can be used as drug carriers, facilitating controlled and targeted drug release to cancer cells [25]. This controlled release can enhance the therapeutic efficacy of anticancer drugs. Surface modification of ZnO nanoparticles can be tailored to enhance their selectivity towards cancer cells. Functionalization with specific ligands or antibodies allows for targeted delivery, reducing off-target effects and minimizing damage to healthy tissues. ZnO nanoparticles are generally considered biodegradable, which can be advantageous for minimizing long-term toxicity concerns [26]. The biodegradation products, primarily zinc ions, are expected to be less toxic and more easily cleared from the body. ZnO nanoparticles can be engineered to possess multifunctional capabilities, including imaging and diagnostics. This can enable simultaneous imaging of cancer cells and targeted therapy, providing real-time monitoring of treatment efficacy [23]. ZnO nanoparticles can be used in combination with other therapeutic modalities, such as chemotherapy or radiotherapy, to achieve synergistic effects. This approach may enhance the overall anticancer efficacy while potentially reducing the required doses of individual treatments. Zinc is an abundant and relatively low-cost material, contributing to the potential cost-effectiveness of ZnO nanoparticles in large-scale production for cancer therapy applications [27].

A notable comprehensive investigation into the anti-tumor properties of ZnO NPs highlighted their ability to selectively target and induce apoptosis in breast cancer cell lines such as MCF-7 and MDA-MB-231 by increasing BAX TNF protein levels [23]. TNF has a well-established role in apoptosis, inflammation and immune response, which are particularly relevant to the anticancer mechanisms of ZnO nanoparticles. TNF is known to have a broad range of effects in cancer biology, including modulating the tumor microenvironment and affecting cancer cell survival, making it a key target for our study. While BAX is a critical pro-apoptotic protein, our primary aim was to investigate the broader immune-modulatory effects of the nanoparticles in the 3D breast cancer model, for which TNF serves as a more comprehensive marker [28,29,30] .

Additionally, when MCF-7 and T47D cell lines were treated with ZnO NPs, a dose-responsive inhibition and apoptotic induction were observed, while normal human embryonic kidney (HEK293) cells remained unaffected [31]. This suggests that ZnO NPs could serve as effective apoptotic agents in the treatment of human breast cancer.

ZnO NPs can be synthesized through chemical, physical, or biological (green syntheses) approaches. In this context, ZnO NPs in our study are synthesized using a green chemistry approach using Zingiber officinale (ginger), leveraging the inherent phytochemicals in ginger, including 6-gingerol, 8-gingerol, 10-gingerol, shogaol, paradols, zingerone, α-curcumene, dehydro-6-gingerdione, 6-shogaol, 6-paradol, 6-dehydrogingerols, 5-acetoxy-6-gingerol, 3,5-diacetoxy-6-gingerdiol and 12-gingerol [32,33,34].

Zingiber officinale, commonly known as ginger, an herb with a long-standing history in traditional medicine, has been identified as a potential biosynthetic source for the generation of ZnO NPs [35]. The Zingiber officinale extract mediate the biosynthesis and confer stability to the nanoparticles. This green synthesis method not only adheres to environmentally sustainable practices by employing water as a solvent and proceeding at room temperature but also enhances the biological compatibility of ZnO NPs due to the phytochemical coating [36–38]. Such an approach mitigates the environmental impact typically associated with nanoparticle production and paves the way for a novel class of biocompatible nanotherapeutics.

Previous studies have demonstrated the synthesis and various biological activities of ZnO nanoparticles, our study is the first to evaluate their anticancer efficacy specifically against a 3D breast cancer model. This model more accurately mimics the tumor microenvironment compared to traditional 2D cell cultures, providing more relevant insights into the therapeutic potential of the nanoparticles. Although antibacterial, anti-inflammatory and other biological activities have been explored in several literatures [39–41], our research uniquely focuses on the anticancer properties of these nanoparticles.

This work aims to develop and characterise Zingiber officinale-derived ZnO NPs and to investigate the antitumor properties of ZnO NPs against a 3D breast cancer spheroid model. This model more accurately mimics the tumor microenvironment in vivo, providing a more predictive platform for evaluating therapeutic efficacy compared to conventional 2D cell cultures. The intricate architecture of 3D spheroids, with their gradient of nutrients, oxygen and cells at different proliferative and quiescent states, offers a formidable structure that often confers resistance to traditional therapeutics [42,43]. By exploring the interactions between these novel nanoparticles and the 3D spheroid model, we endeavor to elucidate the mechanisms of anticancer activity, cytotoxicity and potential pathways of apoptosis and cell cycle arrest instigated by the Zingiber officinale ZnO NPs.

2. Materials & methods

2.1. Ginger root extract preparation

The fabrication of ZnO NPs was executed in two main phases: the extraction of bioactive components from ginger roots and the subsequent synthesis of the nanoparticles [44]. Initially, fresh ginger roots were thoroughly cleaned, diced and pulverized into a fine powder to enhance the extraction efficiency. The powdered ginger (100 g) was then soaked in 1 L of distilled water and incubated at 40°C for 48 h with constant stirring, facilitating the leaching of phytochemicals [45]. Following the extraction, the plant extract was filtered using Whatman filters to yield a clear ginger extract. This extract was then concentrated using a rotary evaporator (BUCHI instruments, Switzerland), gradually decreasing from 300 to 30 rpm at a steady 40°C to procure a dense extract, which was further dried in a fume hood over 72 h to eliminate residual moisture.

2.2. Green synthesis of ZnO NPs

For the nanoparticle synthesis, 1.0 g of zinc acetate (Fisher Scientific, UK) was dissolved in distilled water (50 ml) to form a 0.1 M solution. Concurrently, three dilutions of the dry ginger extract were prepared in distilled water to form 2%, 3% and 4% solutions. Each ginger extract solution was then mixed with the zinc acetate solution and stirred for 4 h at 700 rpm and 60°C. The pH was adjusted to 12 using sodium hydroxide to induce ZnO NP formation. The resultant mixtures were centrifuged at 10,000 rpm for 10 min to isolate the nanoparticles.

The separated NPs were oven-dried at 100°C to ensure complete desiccation.

2.3. Characterization of the Zingiber officinale-derived ZnO NPs

To ascertain the properties essential for their potential anticancer activity, the ginger-ZnO NPs underwent comprehensive characterization. Morphology was examined using scanning electron microscopy (SEM) (Thermo Fischer Scientific, USA), chemical structures were probed through Fourier-transform infrared spectroscopy (FT-IR) (Lambda, USA) and crystallinity was determined via X-ray Diffraction (XRD) (Rigako, Japan).

Dynamic Light Scattering (DLS) analysis using a Nanobrook Omni Zetasizer (Brookhaven, USA) provided insights into the particle size, polydispersity index (PDI) and zeta potential, which reflects particle surface charge and stability. The size distribution was gauged at a 0.2 mg/ml concentration in distilled water, while zeta potential measurements were conducted in triplicate to ensure reliability.

FT-IR spectroscopy, performed at room temperature using a Lambda Scientific FTIR 7600 with an attenuated total reflection ZnSe crystal, identified functional groups by scanning from 400 to 4000 cm^-1. This range, encapsulating most of the infrared spectrum, allowed for a detailed analysis of the ZnO NPs' chemical bonds and functional groups.

XRD analysis utilized the Rigaku Miniflex 600 benchtop system (Japan), leveraging CuKα radiation at specific settings (40 kV and 15 mA), optimized to acquire precise crystalline structure data. The diffraction patterns, collected across a 2θ range typical for zinc oxide, were interpreted using SmartLab Studio II software, which provided detailed information on the atomic structure and crystallinity.

SEM imaging required sputter-coating the nanoparticles with gold to enhance conductivity, performed under vacuum at an accelerating voltage of 15 kV. The Phenom XL G2 microscope revealed the NPs' morphology, while Energy Dispersive X-Ray (EDX) spectroscopy, conducted with an AXS EDS system, confirmed their elemental composition, verifying the presence of zinc and oxygen and ruling out contaminants.

2.4. Assessment of ZnO NPs antiproliferative activity against 2D MCF-7 cell line

The antiproliferative efficacy of the synthesized ZnO NPs was evaluated using the MTT cell viability assay. For the assay, 3 × 10³ MCF-7 cells (ATCC HTB-22^™) were seeded into each well of a 96-well plate and treated in triplicate for 72 h with varying concentrations of ZnO NPs, with the final volume in each well brought up to 200 μl. Control wells contained untreated cells and cells treated with 1% DMSO as negative controls to establish a baseline response. At the designated time-point, 0.05 mg/ml of MTT reagent (Thermo Fischer Scientific, USA) was added to each well and the cells were further incubated for 4 h, allowing for the accumulation of formazan within viable cells. Subsequently, the medium containing MTT was carefully removed and the formazan crystals were dissolved in 150 μl of DMSO. The absorbance was measured at 590 nm using a BioTek Cytation 5 multi-mode plate reader (USA). A reference wavelength of 630 nm was used to correct for background absorbance. The IC₅₀ value was determined by plotting the concentration of nanoparticles against the percentage of viable cells and fitting the data to a dose-response curve using nonlinear regression analysis [46].

2.5. Generation of 3D breast cancer model & the anti-tumor effects of ZnO NPs

The hanging drop technique was used to generate 3D spheroids from MCF-7 breast cancer cell line. For spheroid generation, a cell suspension containing 25 × 10⁴ cells in 1 ml of culture media supplemented with 1% D-glucose was prepared. From this suspension, 20 μl droplets were pipetted onto the inner lid of a 6-well plate. To prevent evaporation, the plate was carefully inverted and the droplets were incubated for 72 h to allow for spheroid formation. Following this incubation period, the spheroids were transferred to a 6-well plate coated with Matrigel (Corning, USA) and allowed to adhere. The size and morphology of the spheroids were meticulously monitored over a 14-day period using an inverted microscope. After spheroid formation, they were relocated to a 96-well plate for treatment assays. Spheroids were exposed to various concentrations of ZnO NPs and the treatments were performed in triplicate. The incubation continued for another 14 days. The spheroids' volume was quantitatively analyzed using MOTIC Image Plus Software (China). The efficacy of the treatments was expressed as a percentage inhibition of spheroid volume when compared with untreated control groups. This metric provided a clear indication of the antiproliferative effects of the ZnO NPs on cancer cell spheroids.

2.6. ELISA for TNF-α expression

To assess the inflammatory response to ZnO NPs, MCF-7 cells were exposed to 50 and 100 μg/ml concentrations and incubated for 72 h. ELISA was then performed on the supernatants to measure TNF-α levels using human TNF-α ELISA kit (Abcam, UK). The process involved adding the supernatant and an antibody cocktail to a 96-well plate, followed by a 1-hour incubation with shaking. After washing, a TMB substrate was added, the reaction was stopped post-15 min and the optical density was measured at 450 nm. TNF-α concentrations were determined against a standard curve.

2.7. Statistical analysis

Statistical analysis was conducted using GraphPad Prism version 9 software via one-way ANOVA. p values of less than 0.05 were considered statistically significant.

3. Results & discussion

Nanotechnology has been demonstrating transformative potential across multiple scientific domains [13,47]. One of the areas that has benefited immensely from the advancements in nanotechnology is cancer therapy [45,48]. Traditional cancer treatment modalities, including chemotherapy, radiation and surgery, while effective, often come with a multitude of challenges, such as non-specificity, collateral damage to healthy tissues and debilitating side effects [49,50]. Nanotechnology, in its essence, seeks to surmount these challenges by offering solutions that are targeted, efficient and potentially less detrimental [51,52].

While the promise of nanotechnology is unquestionable, the methods employed in the synthesis of nanoparticles are of paramount significance [53]. The conventional chemical and physical methods, although effective, are often energy-intensive, use toxic solvents and might generate hazardous by-products. To address these concerns and to tread on a path of sustainability, the green synthesis of metallic nanoparticles has emerged as a compelling alternative [54].

Green synthesis capitalizes on environmentally benign agents, such as plant extracts, microorganisms and enzymes, to reduce and stabilize metallic ions, culminating in the formation of metallic nanoparticles [55]. This eco-friendly approach not only negates the requirement for hazardous chemicals but also operates at ambient conditions, making it energy efficient. For instance, plant-derived phytochemicals, owing to their abundance in reducing agents, have been exploited to synthesize a plethora of metallic nanoparticles, such as silver [56], gold [57] and zinc oxide [58]. The biogenic synthesis route not only epitomizes an eco-friendly approach but also ensures the production of biocompatible nanoparticles, a trait indispensable for medical applications [59].

Among the myriad of nanoparticles being investigated for therapeutic applications, zinc oxide nanoparticles (ZnO NPs) hold a special place, primarily due to their multifaceted benefits. ZnO NPs exhibit exceptional physicochemical properties, such as high catalytic activity, UV absorption and photoluminescence, making them suitable for various applications ranging from electronics to cosmetics. However, it's their therapeutic potential that stands out [60,61].

In the realm of oncology, ZnO NPs have shown promise due to their inherent ability to induce selective cytotoxicity towards cancerous cells. Several studies have demonstrated that ZnO NPs can induce reactive oxygen species (ROS) generation in malignant cells, leading to oxidative stress-mediated apoptosis. This mechanism, combined with their ability to release zinc ions, which can interfere with cellular processes, makes ZnO NPs potent anti-cancer agents [23,27].

Furthermore, their biocompatibility, combined with their potential for surface modifications, enables the development of targeted drug delivery systems. Coating ZnO NPs with specific ligands or antibodies ensures their directed delivery to tumors sites, thereby augmenting therapeutic outcomes while minimizing off-target effects [60,62]. Additionally, their semiconductor nature makes them suitable for photodynamic therapy, where they can be activated using UV radiation to generate ROS, further amplifying their anti-cancer effects [25].

In the ever-evolving realm of nanotechnology, this study aimed to conjoin the promising attributes of ZnO NPs with the inherent bioactive potential of Zingiber officinale, more commonly known as ginger [44]. Ginger, revered in traditional medicine and acknowledged for its plethora of phytochemicals, served as the green reducing agent in the synthesis of ZnO NPs. By employing Zingiber officinale, this study not only leaned towards an eco-friendly synthesis approach but also potentially imparted the therapeutic attributes of ginger to the nanoparticles [63,64].

The role of ginger extract is primarily attributed to its bioactive compounds, which act as reducing and capping agents. Ginger (Zingiber officinale) is known to contain various phytochemicals, such as flavonoids, phenolic compounds and terpenoids, which possess reducing properties. These compounds play a crucial role in the conversion of zinc ions to zinc oxide nanoparticles while also stabilizing and controlling the size of the nanoparticles [65].

The bioactive compounds in ginger extract, particularly the phenolic compounds and flavonoids, can donate electrons. During the green synthesis process, these compounds act as reducing agents, facilitating the reduction of zinc ions (Zn²⁺) to form zinc nanoparticles (ZnO). The reduction step is a key aspect of nanoparticle formation in green synthesis methods [65].

The same bioactive compounds that act as reducing agents also serve as capping agents. Capping agents play a crucial role in preventing the agglomeration or clustering of nanoparticles. They form a protective layer around the nanoparticles, stabilizing them and preventing uncontrolled growth or aggregation. This is important for obtaining well-dispersed and stable nanoparticles [66].

The bioactive compounds in ginger extract contribute to the control of the size of the synthesized nanoparticles. The concentration and type of phytochemicals present in the ginger extract can influence the size distribution of the resulting nanoparticles. Proper size control is essential for achieving desired properties and applications of the zinc oxide nanoparticles [65]. Characterization of nanoparticles is crucial to understand their physicochemical attributes and predict their behaviour in biological systems. In this context, this study employed zeta sizer to decipher the hydrodynamic size, charge (zeta potential) and polydispersity index (PDI) of the synthesized ZnO NPs [67]. The size of nanoparticles is pivotal in determining their cellular uptake, biodistribution and eventual fate in the body. Additionally, the zeta potential provides insight into the stability of the nanoparticle suspension, with values further from zero indicating enhanced colloidal stability due to repulsive forces between particles [68]. Meanwhile, the PDI, indicative of the size distribution, offers clues about the homogeneity of the nanoparticles. A low PDI is often desired as it signifies a uniform population, leading to predictable in vivo behaviour [69].

3.1. Hydrodynamic size, polydispersity & zeta potential analysis of ZnO NPs

Using a Nano Zeta-sizer, the synthesized ZnO NPs were characterized for hydrodynamic size, polydispersity index (PDI) and surface charge. The dynamic light scattering (DLS) results, as shown in Table 1, indicated average diameters of 188.9, 175.7 and 171.2 nm for ZnO NPs at 2, 3 and 4% concentrations, respectively, suggesting smaller particle formation at higher concentrations. PDI values were all below 0.2, showing a uniform size distribution for each concentration. Zeta potential measurements, indicative of particle stability, showed -8.2, -11.7 and -9.7 mV for the respective concentrations, demonstrating good stability across the formulations.

Table 1.

Characterisation of size, PDI and zeta potential of the Zingiber officinale-derived ZnO NPs.

Formula	2%	3%	4%
Diameter (nm)	188.9 ± 6.76	175.7 ± 9.21	171.2 ± 8.37
PDI	0.057 ± 0.02	0.119 ± 0.044	0.168 ± 0.057
Zeta potential (mV)	-8.2 ± 3.1	-11.7 ± 4.8	-9.7 ± 4.1

Throughout the 30-day observation period, all three ZnO NP formulations demonstrated stable particle size. There were no discernible significant fluctuations in the diameter of the nanoparticles for any of the formulations, suggesting that the particles resisted aggregation and maintained their size integrity over time. Consistent with the size data, the PDI values for all formulations remained below 0.2 throughout the one-month study duration. The consistent low PDI values indicate a uniform nanoparticle size distribution, further reinforcing the notion that no significant agglomeration or morphological changes took place in the samples during the test period.

The zeta potential is a crucial parameter that reflects the surface charge of nanoparticles and can indicate colloidal stability. Throughout the assessment period, all formulations maintained a stable zeta potential. This continuous stability in zeta potential indicates a strong repulsion between individual nanoparticles, suggesting that they are unlikely to aggregate over time (Figure 1).

Figure 1.

Assessment of ZnO NPs stability. ZnO NPs formulations stability was assessed over a 28-day at room temperature in terms of size, PDI and zeta potential.

3.2. FTIR analysis

FTIR spectroscopy was utilized to characterize the ZnO NPs synthesized with 2, 3 and 4% ginger root extract concentrations. ZnO NPs have been prepared with different concentrations of *Zingiber officinale* extract (2, 3 and 4%). FTIR analysis was performed to investigate various surface properties and interactions to compare the effect of different extract percentages and investigate the impact of varying extract concentrations on the surface chemistry and functional groups present on the ZnO nanoparticles and to establish a comprehensive understanding of the interaction between the extract and ZnO nanoparticles.

The FTIR spectra exhibited consistent peaks across all concentrations, confirming the presence of ZnO in the synthesized particles (Figure 2). The peak around 430 cm^-1, present in all three samples, is indicative of the Zn-O stretching vibrations, which is characteristic of ZnO NPs. The broad band observed near 3430 cm^-1 points to O-H stretching vibrations from physically adsorbed water on the NPs' surface and the peak at 1630 cm^-1 is attributed to the bending vibrations of water molecules.

Figure 2.

FTIR analysis of ZnO NPs prepared using different concentrations of ginger root extract: (A) for 2%, (B) for 3% and (C) for 4%. [The peak around 430 cm^-1, present in all three samples, is indicative of the Zn-O stretching vibrations, which is characteristic of ZnO NPs. The broad band observed near 3430 cm^-1 points to O-H stretching vibrations from physically adsorbed water on the NPs' surface and the peak at 1630 cm^-1 is attributed to the bending vibrations of water molecules].

Additionally, the presence of a peak at 914 cm^-1 suggests C-H bending from organic compounds, likely remnants of the ginger extract on the ZnO NP surface. Peaks around 1000 cm^-1 support the existence of metal-oxygen bonds, reinforcing the formation of Zn-O.

While the zeta sizer provided essential information, the comprehensive characterization of ZnO NPs necessitated the employment of additional techniques. FTIR was used to identify the functional groups on the nanoparticle surface, which can shed light on the interaction between ginger's phytochemicals and the ZnO matrix. According to literature regarding FTIR spectrum, you would typically observe characteristic peaks corresponding to different vibrational modes of the chemical bonds present in zinc oxide nanoparticles. General features observed at the most prominent peaks are usually associated with the stretching vibrations of Zn-O bonds. These peaks are typically observed in the range of 400–600 cm^-1. Peaks in the 200–400 cm^-1 range are often attributed to bending modes of Zn-O bonds. Presence of hydroxyl groups might be indicated by peaks in the range of 3000–3600 cm^-1. Broad peaks around 1600–1800 cm^-1 may indicate the presence of adsorbed water on the surface of nanoparticles [70]. Peaks in the 1600–1700 cm^-1 range could suggest the presence of zinc hydroxide species. The FTIR spectra of the synthesized ZnO nanoparticles revealed several prominent peaks, the most significant peak was identified at approximately 430 cm^-1, this peak was attributed to the Zn-O stretching vibrations, confirming the formation of ZnO nanoparticles. Additionally, a broad peak at around 3430 cm^-1 was observed. This peak corresponds to O-H stretching vibrations, suggesting the presence of physically adsorbed water on the nanoparticle surfaces [71]. Furthermore, a strong absorption peak near 1630 cm^-1 was associated with the bending vibrations of H-O-H, indicating the presence of water molecules on the nanoparticles. Weak signals were noted at other spectra. A peak at 914 cm^-1 was observed in the FTIR spectra, corresponding to the C-H bending of alkenes. This observation implies the presence of organic molecules, likely from the ginger root extract, on the surface of the synthesized ZnO nanoparticles. Bands found near 1,000 cm^-1 were associated with metal-oxygen tension and bending, further supporting the formation of Zn-O bonds in the nanoparticles. No significant peaks corresponding to impurities were found in the spectra, indicating the high purity of the synthesized ZnO nanoparticles [72]. Additionally, the lack of peaks linked to other zinc or oxygen compounds in the formulations further confirms the successful synthesis of ZnO nanoparticles.

3.3. SEM & EDX Evaluation of ZnO Nanoparticles

ZnO nanoparticles formulated with 2, 3 and 4% ginger root extract concentrations were analysed for morphology and elemental composition using SEM and Energy Dispersive X-Ray EDX analysis. SEM images depicted spherical nanoparticles for all formulations with noticeable agglomerations, likely due to the drying effects during sample preparation (Supplementary Figure S1).

The EDX analysis confirmed the primary presence of zinc and oxygen, with minor impurities of carbon and chloride noted (Supplementary Figure S2). The consistent morphology and composition across varying concentrations attest to the effective synthesis of ZnO nanoparticles via the green chemistry approach utilizing ginger extract.

SEM and EDX were pivotal in visualizing the morphology of the nanoparticles and understanding their elemental composition [60,73]. Such information can correlate with the nanoparticles' biological activity, as shape and elemental attributes can influence cellular interactions.

3.4. XRD characterization of ZnO NPs

XRD analysis was conducted on ZnO nanoparticles synthesized using 2, 3 and 4% ginger root extract concentrations to ascertain their crystalline structure and purity. The XRD patterns (Supplementary Figure S3) exhibited peaks at 2θ values of 31.9, 34.55, 36.35, 47.65 and 56.70°. These peaks are indicative of the wurtzite hexagonal phase of ZnO and align with the planes (100), (002), (101), (102) and (110) as identified by the JCPDS card No. 36-1451.

The most pronounced peak across all formulations was at the (101) plane, suggesting this orientation's dominance in the crystalline structure. The absence of impurity-related peaks in the XRD patterns confirmed the high purity of the nanoparticles.

Crystallite sizes, derived using the Scherrer equation, were calculated to be 45.6 nm, 44.63 nm and 42.23 nm for the 2%, 3% and 4% extract formulations, respectively. This indicates that the ZnO nanoparticles synthesized with the 2% extract formulation exhibit the highest crystallinity due to their largest crystallite size, followed by the 3% and 4% formulations with progressively smaller crystallite sizes. The variance between sizes observed via DLS and XRD is attributed to DLS measuring the hydrodynamic size, which encompasses the particle and its surrounding solvent shell, while XRD assesses the size of the crystalline domains within the particles, offering a smaller measurement. The hydration layer and potential particle aggregation during DLS, versus the crystallite-specific data from XRD, explain the differences in size measurements reported by these two analytical techniques.

XRD was employed to ascertain the crystalline nature of the nanoparticles. The crystal structure and phase purity of ZnO NPs can influence their photocatalytic and therapeutic properties [74]. In XRD patterns, diffraction peaks correspond to the arrangement of atoms within the crystal lattice, allowing researchers to identify the crystal structure and estimate the size of nanoparticles. For zinc oxide nanoparticles, we expect to observe characteristic diffraction peaks associated with its crystalline phases [75]. According to literature the most common crystalline structures of ZnO are hexagonal wurtzite and cubic zincblende. The hexagonal wurtzite structure is more stable for ZnO at room temperature. Typically, the major diffraction peaks for ZnO in the hexagonal wurtzite structure can be found at 2θ values around 31.8°, 34.4°, 36.2°, 47.6°, 56.5°, 62.8° and 67.9°. These peaks correspond to the (100), (002), (101), (102), (110), (103) and (112) crystallographic planes, respectively. It's important to note that the exact positions of the peaks can vary depending on factors such as the size of the nanoparticles, strain and possible impurities. The broadening of the peaks is often observed in nanoparticle samples due to the finite size effect [76,77].

The XRD patterns obtained for the formulations reveled that the diffraction peaks observed at 2θ values of 31.9°, 34.55°, 36.35°, 47.65°, 56.70 were characteristic of the wurtzite hexagonal phase of ZnO, which are correspondingly assigned to the (100), (002), (101), (102) and (110) planes respectively, as per the standard Joint Committee on Powder Diffraction Standards (JCPDS) card No. 36-1451 [75,78]. For all formulations, the intensity of the (101) peak was found to be the highest, suggesting that the synthesized ZnO nanoparticles predominantly had a (101) crystal plane orientation. Additionally, the absence of other impurity peaks confirmed the high purity of the synthesized ZnO nanoparticles. Actual XRD patterns may vary depending on the synthesis method, conditions and specific properties of the zinc oxide nanoparticles [79,80].

The calculated crystallite sizes, using the Scherrer equation, were found to be 45.6 nm, 44.63 nm and 42.23 nm for the 2%, 3% and 4% formulations respectively. The difference in particle sizes determined by XRD compared to that of DLS analysis is not uncommon and can be attributed to the different principles on which these two techniques operate. DLS determines the hydrodynamic diameter of particles. This includes not only the actual particle itself but also the “solvent shell” or “hydration layer” around the particle, leading to a larger observed size. On the other hand, XRD is used to determine the size of the crystalline domains within the particles (also referred to as crystallite size), which will always be smaller than or equal to the actual particle size. This is because a single nanoparticle could be composed of one or more crystallites. Therefore, the larger size observed in DLS compared to XRD for ZnO NPs can be attributed to the hydration layer and possible aggregation of NPs in the DLS measurement, while the XRD gives the size of the individual crystalline domains.

When contextualized within the vast expanse of literature, the findings of this study resonate with multiple precedents. Numerous studies have vouched for the therapeutic potential of ZnO NPs synthesized using ginger root extract. However, the size of the synthesized particles significantly varied among these studies from 30–320 nm [81,82].

3.5. The antiproliferative effects of ZnO NPs on the 2D MCF-7 breast cancer cells

The antiproliferative potential of ZnO NPs on the conventional 2D MCF-7 breast cancer cells were assessed after their successful synthesis and characterization. MCF-7 cells were treated with varying ZnO NP concentrations (2%, 3% and 4%) ranging from 1.56 to 200 μg/ml. The results showed IC₅₀ values of 14.7 μg/ml, 26.2 μg/ml and 47 μg/ml for the 2, 3 and 4% ZnO NP formulations, respectively (Figure 3). The 2% ZnO NP formulation demonstrated the highest cytotoxicity with the lowest IC₅₀ value, signifying its superior efficacy in inhibiting MCF-7 cell proliferation.

Figure 3.

Dose-response curves for MCF-7 breast cancer cells treated with varying concentrations of ZnO NPs formulations of the ginger root extract: (A) for 2%, (B) for 3% and (C) for 4%. Data represent mean ± SD. [MCF-7 cells were treated with varying ZnO NP concentrations (2, 3 and 4%) ranging from 1.56 to 200 μg/ml. The results showed IC₅₀ values of 14.7, 26.2 and 47 μg/ml for the 2%, 3% and 4% ZnO NP formulations, respectively. The 2% ZnO NP formulation demonstrated the highest cytotoxicity with the lowest IC₅₀ value, signifying its superior efficacy in inhibiting MCF-7 cell proliferation].

3.6. Antiproliferative effects of ZnO NPs on the 3D MCF-7 spheroids

The MCF-7 spheroid model offers a more representative biological context for evaluating anticancer agents compared to 2D cultures due to its mimicry of the tumor microenvironment [83]. The 2% ZnO NP formulation, identified as the most potent through MTT assays, was further assessed on MCF-7 spheroids using concentrations of 50, 100 and 200 μg/ml. A clear dose-dependent reduction in spheroid viability was observed with the ZnO NPs (Figure 4). Notably, there was a significant 65.17% reduction in spheroid size at 50 μg/ml and a complete disintegration at 100 and 200 μg/ml concentrations, indicating a potent antiproliferative effect at higher doses.

Figure 4.

Dose-dependent inhibitory effects of the 2% ZnO NP formulation on MCF-7 spheroid model. [The MCF-7 spheroid model offers a more representative biological context for evaluating anticancer agents compared to 2D cultures due to its mimicry of the tumor microenvironment. The 2% ZnO NP formulation, identified as the most potent through MTT assays, was further assessed on MCF-7 spheroids using concentrations of 50, 100 and 200 μg/ml. A clear dose-dependent reduction in spheroid viability was observed with the ZnO NPs. Notably, there was a significant 65.17% reduction in spheroid size at 50 μg/ml and a complete disintegration at 100 and 200 μg/ml concentrations, indicating a potent antiproliferative effect at higher doses].

The antiproliferative effects of ZnO nanoparticles (NPs) on 3D MCF-7 spheroids refer to the study of how these nanoparticles impact the growth and proliferation of breast cancer cells (MCF-7) when organized in three-dimensional (3D) spherical structures [84]. The differential response between MCF-7 and HT-29 cells towards the ZnO NPs is intriguing. Previous research has also reported varied sensitivities of different cancer cells to ZnO NPs. Such differential behavior emphasizes the heterogeneous nature of cancer and the need to personalize therapeutic strategies. The greater resistance of HT-29 cells could be attributed to their distinct cellular uptake mechanisms, genetic makeup, or defense mechanisms [23].

Our findings illustrated a significant susceptibility to ZnO NP treatment, with the 2% formulation exhibiting the most potent cytotoxicity. This corroborates with existing literature, where several studies have expounded on the preferential toxicity of ZnO NPs towards breast cancer cells, mediated through reactive oxygen species generation and apoptosis induction. For instance, a studconducted by George et al. (2022) revealed that ZnO NPs exhibited a strong anti-proliferative effect against MCF-7 by inducing ROS and apoptosis [85]. Similarly, Boroumand Moghaddam et al. (2017) reported an IC₅₀ of 121 μg/ml against the MCF-7 cell line after 24 h of treatment with the ZnO NPs [84]. The lower IC₅₀ reported in this thesis is largely attributed to the longer time-point used during this study.

Spheroids represent three-dimensional (3D) multicellular aggregates that are cultivated in vitro, aimed at stimulating the intricate and multifaceted cellular microenvironment present within solid tumors in vivo [42].

The relevance of spheroids is bolstered by the growing acknowledgment of the limitations of 2D cultures in truly capturing tumors biology. Spheroids, with their 3D architecture, offer a more faithful representation of in vivo tumors conditions such as cell-to-cell interactions, matrix deposition, differential proliferative indices and therapeutic resistance [86,87]. Thus, they serve as a crucial bridge between in vitro experiments and in vivo animal models, allowing for a more accurate prediction of how cancer cells might respond to therapeutic agents in the human body [88].

A review of the existing literature highlights a limited number of studies investigating the impact of ZnO NPs on 3D spheroid models. However, a noteworthy investigation led by Wang et al. (2017) reported that ZnO NPs not only potentiated the cytotoxic effects of doxorubicin against the multi-drug resistant breast cancer cell line, MDA-MB-231, but also showcased their potential as pH-dependent drug nanocarriers. This enhancement was reflected in heightened cellular uptake and augmented penetration into tumor spheroids [89]. Complementing this observation, another significant study by Chabattula et al. (2021) delineated the dose-responsive cytotoxic nature of ZnO NPs when administered to lung cancer A549 cells. This was manifestly demonstrated by a pronounced reduction in the size of the A549 spheroid models [90]. Such revelations from pioneering works offer insightful perspectives, anchoring our understanding and lending credence to the findings of the present study.

3.7. TNF-α expression induced by ZnO NP treatment

Investigating the cytotoxic mechanisms of ZnO NPs on MCF-7 cells, this study focused on the inflammatory response as a possible pathway, particularly the expression of tumor Necrosis Factor-alpha (TNF-α). Elevated TNF-α levels often signify cellular stress and inflammation, which could contribute to the antiproliferative effects observed. Untreated MCF-7 cells showed a baseline TNF-α level of 22.43 pg/ml. Post-treatment with ZnO NPs, TNF-α expression significantly increased to 62.2 pg/ml at 50 μg/ml and 93.6 pg/ml at 100 μg/ml of ZnO NP concentration (Figure 5). These results highlight an inflammatory response induced by ZnO NPs and suggest that such inflammation could be a contributing factor to the nanoparticles' cytotoxicity in breast cancer cells.

Figure 5.

Analysis of TNF-α expression in MCF-7 cells following treatment with the 2% formula of ZnO NPs. *p value < 0.05 and **p < 0.01 calculated by one-way ANOVA. Data represent mean ± SD (n = 3) [Untreated MCF-7 cells showed a baseline TNF-α level of 22.43 pg/ml. Post-treatment with ZnO NPs, TNF-α expression significantly increased to 62.2 pg/ml at 50 μg/ml and 93.6 pg/ml at 100 μg/ml of ZnO NP concentration].

TNF-α is a pro-inflammatory cytokine and an essential mediator of the inflammatory response [91]. Its role in a variety of cellular processes such as cell proliferation, differentiation and apoptosis has been extensively studied. Notably, while moderate levels of TNF-α can drive cell proliferation and survival, overexpression often leads to inflammation-driven apoptosis and cell death [92,93].

In the current study, treatment of MCF-7 cells with ZnO NPs led to a significant upregulation of TNF-α levels, correlating with the observed cytotoxic effects in the MTT assay and spheroid evaluations. This highlights the idea that the cytotoxicity of ZnO NPs could be mediated, at least in part, through an inflammatory response. ZnO NPs, like other metal-based nanoparticles, are known to elicit immune responses, manifesting as upregulation of pro-inflammatory mediators [94,95].

The literature has furnished evidence suggesting that ZnO NPs can stimulate the production of ROS which, in turn, activates various cellular pathways, one of which results in the upregulation of inflammatory cytokines including TNF-α [96–98]. Moreover, ZnO NPs was reported to induce oxidative stress in human epidermal cells, leading to increased TNF-α and interleukin-8 levels [96]. Furthermore, several other research undertakings have elucidated that ZnO NPs can initiate the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, a primary inflammatory signaling cascade. The result is the transcription of various pro-inflammatory genes, including TNF-α [99–101].

The use of a 3D breast cancer spheroid model accurately replicates the tumor microenvironment, offering more predictive insights into therapeutic efficacy compared to conventional 2D cultures. This model better mimics the gradients of nutrients, oxygen and cellular states present in actual tumors. Moreover, employing Zingiber officinale-infused ZnO nanoparticles synthesized through a green synthesis approach. This method not only offers an eco-friendly alternative but also introduces bioactive compounds into the nanoparticles that could potentially enhance their anticancer activity. Furthermore, by studying the interactions between these novel nanoparticles and the 3D spheroid model, we provide insights into their anticancer mechanisms, including cytotoxicity, apoptosis induction and effects on cell cycle regulation. Finally, this approach could lead to breakthroughs in therapeutic efficacy and safety.

4. Conclusion

This is the first research that examines at the cytotoxic effects of ZnO NPs derived from Zingiber Officinale on colorectal and MCF-7 spheroid model cell lines. It showed substantial anticancer activity against MCF-7 cells, indicating a possible inflammatory response via elevated TNF-α levels. These findings show that ZnO NPs might be interesting candidates for targeted cancer therapeutics, necessitating more biological research to investigate their mechanisms of action and therapeutic potential.

Future study should focus on manufacturing ZnO NPs using different green technologies and comparing their effectiveness and biocompatibility. Increasing the number of cancer cell lines investigated will aid in assessing the nanoparticles' overall activity. Furthermore, in vivo evaluations will be required to translate in vitro findings into practical applications. Understanding the processes of interaction between these nanoparticles and cells, as well as scale-up research for large-scale manufacture, will be critical for furthering their application in cancer therapies.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/20565623.2024.2419806

Author contributions

R Alhaddad, BM Abualsoud, I Al-Deeb and H Nsairat all contributed in similarly to this work through surveying, searching, data collection, experiments, results discussion, writing and finalizing the final draft.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.