Anticancer Activity of Zinc Oxide Nanoparticles Biosynthesized Using Urtica pilulifera L. Extract Against U87 Glioblastoma Cancer Cells

INTRODUCTION
Cancer remains a formidable global health challenge, characterized by aggressive cellular proliferation and metastatic potential, which often lead to poor clinical outcomes. The complexity of the tumor microenviro, nment and the innate ability of malignant cells to evade programmed cell death (apoptosis) make it a leading cause of mortality worldwide [1]. Among the most lethal forms of malignancy is Glioblastoma Multiforme (GBM), represented by the U87 cell line, which is notorious for its rapid progression and resistance to standard interventions [2]. While conventional therapies such as chemotherapy and radiotherapy are widely utilized, their clinical efficacy is frequently compromised by severe systemic toxicity, non-specific targeting of healthy tissues, and the rapid emergence of multi-drug resistance (MDR) [1, 2, 3]. These therapeutic hurdles underscore the urgent need for innovative, highly selective, and biocompatible anticancer agents that can deliver maximum lethality to tumor cells with minimal collateral damage to normal physiological systems [3].
In response to these challenges, recent biomedical research has pivoted toward nanotechnology to develop “smart” therapeutic modalities. Nanoparticles (NPs), due to their unique physicochemical propertiesincluding a high surface-area-to-volume ratio and enhanced cellular permeabilityoffer a superior platform for targeted drug delivery and intrinsic cytotoxicity [4]. Among various inorganic nanomaterials, zinc oxide nanoparticles (ZnONPs) have gained significant prominence in oncology. ZnONPs are recognized for their inherent ability to induce selective apoptosis in malignant cells via the generation of reactive oxygen species (ROS), which triggers oxidative stress and mitochondrial dysfunction [4, 5]. Furthermore, their pH-sensitive nature allows for the accelerated release of Zn{2+} ions in the acidic microenvironment of tumors, further enhancing their selective toxicity while maintaining a favorable safety profile for healthy tissues [5].
Despite the therapeutic potential of ZnONPs, traditional synthesis routes involving physical and chemical methods often present significant environmental and biological drawbacks. These processes frequently utilize hazardous reducing agents, such as sodium borohydride, and require high energy consumption, leading to the production of toxic by-products [6]. To address these sustainability concerns, “green synthesis” has emerged as an eco-friendly and cost-effective alternative. This approach utilizes biological materialsspecifically plant extractsto serve as both reducing and stabilizing agents in a single-step reaction [6, 8]. Green-synthesized nanoparticles often exhibit superior biocompatibility and enhanced biological activity due to the natural “capping” of bioactive molecules on their surface, which prevents aggregation and improves stability [8].
In this context, Urtica pilulifera (Roman Nettle) represents an ideal biological scaffold for nanoparticle fabrication. This medicinal plant is exceptionally rich in secondary metabolites, including phenolic acids, flavonoids, tannins, and terpenoids, which are known for their potent antioxidant and reducing capabilities [7]. These phytochemicals facilitate the efficient reduction of zinc precursors into stable nanoparticles while providing a natural layer of bioactive compounds that may synergistically enhance the anticancer effects [7, 8]. Although the general properties of ZnONPs are well-documented, the specific interaction between U. pilulifera-mediated nanoparticles and aggressive brain cancer models like U87 glioblastoma remains an area that requires comprehensive investigation to optimize dose-dependent responses and therapeutic outcomes.
This study aims to synthesize zinc oxide nanoparticles using an aqueous extract of U. pilulifera via a one-pot, eco-friendly green approach. Following synthesis, the physicochemical features of the produced ZnONPs including their morphology, crystallinity, and size distribution are characterized using advanced analytical instruments such as SEM, XRD, and UV-Vis spectroscopy. Finally, the research assesses the anticancer activity and dose-dependent effects of these synthesized ZnONPs against U87 glioblastoma cells in vitro. By exploring the cytotoxic thresholds and the induction of apoptosis in these resistant cells, this work seeks to provide new insights into the development of plant-mediated Nano medicines as a potent and selective tool for targeted cancer therapy.

MATERIALS AND METHODS
Plant Collection and Extraction
Fresh leaves of U. pilulifera were collected from uncontaminated sites and transported to the laboratory in sterile containers to prevent microbial contamination. The leaves were thoroughly washed using distilled water to remove dust and debris, air-dried under shade to preserve heat-sensitive phytochemicals, and then ground into a fine powder using an electric mill. For aqueous extraction, 50 g of powdered material were mixed with distilled water and heated at 70 °C for 30 minutes to facilitate the release of bioactive compounds [9]. The mixture was filtered through Whatman No.1 paper, and the filtrate was stored at 4 °C for subsequent nanoparticle synthesis.

Phytochemical Screening
Qualitative phytochemical tests were performed to identify major classes of bioactive compounds present in the extract. Standard methods confirmed the presence of phenolics, flavonoids, tannins, alkaloids, terpenoids, and coumarins, all of which are known to possess strong reducing and antioxidant activities that support nanoparticle formation and stabilization [10].

Green Synthesis of ZnONPs
To prepare zinc oxide nanoparticles, the aqueous plant extract was added dropwise to a 1 mM zinc nitrate solution under continuous magnetic stirring. The appearance of a cloudy or milky-white dispersion indicated the initiation of nanoparticle formation. The reaction mixture was incubated in the dark for 24 hours to prevent photoactivation of zinc ions [11]. The resulting suspension was centrifuged at 10,000 rpm for 15 minutes, washed with distilled water three times to remove impurities, and then dried to obtain purified ZnONPs.

Characterization of Nanoparticles
The synthesized ZnONPs were characterized using several instrumental techniques: These included FE-SEM for morphological examination, UV-Vis spectroscopy for assessing the crystalline properties, XRD for structural analysis, and Fourier transform infrared spectroscopy (FTIR).

Cell Culture (U87 Human Glioblastoma Cell Line)
U87 cells were cultured in MEM medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified incubator. The MTT assay was used to estimate cytotoxicity after treating the cells with different concentrations of U-ZnONPs (0–0.01 µg/ml) for 48 hours. MTT solution was then added, followed by DMSO, to dissolve the formazan, and absorbance was measured at 492 nm.

Cytotoxicity ratio = [(ODcontrol–ODsample)/ODcontrol] × 100

Morphological Examination
Cell morphological changes after treatment with U-ZnONPs were observed under an inverted light microscope. Parameters such as cell shrinkage, loss of adherence, and membrane blebbing were recorded as indicators of apoptosis.

Statistical Analysis
The results were statistically analyzed based on the completely randomized design, the least significant difference (LSD) test, and the Duncan test for reproducibility between means at the probability level (p≤0.05).

Ethical Considerations
The project was approved as a service evaluation and registered with the Department of Ecology within the department’s science plan. Formal ethical review was waived in accordance with institutional policy, as the project included routine laboratory data without specification. The authors declared no conflict of interest.

Reporting Standards
This quality improvement project was prepared in accordance with the SQUIRE 2.0 Excellence in Quality Improvement Reporting Standards guidelines [17]. The SQUIRE framework is specifically designed to guide the reporting of systematic efforts to improve the quality, safety, and value of healthcare. A complete SQUIRE checklist is available in the supplementary materials accompanying this manuscript.

RESULTS AND DISCUSSION
Detection of active components in U. pilulifera plants
Table 1 presents the phytochemical screening results of the aqueous extract of U. pilulifera, revealing the presence of several bioactive compounds. Positive results were obtained for terpenes, tannins, terpenoids, flavonoids, phenols, coumarins, and alkaloids. In contrast, the extract tested negative for glycosides, resins, and saponins. 

Identification of U-ZnON
Morphological and Structural Analysis (SEM & XRD)
The surface topography and grain characteristics of the synthesized nanoparticles were evaluated using Field Emission Scanning Electron Microscopy (FE-SEM). The micrographs (Fig. 1) reveal a predominantly spherical morphology, where primary nanoparticles tend to form clusters or agglomerates. This phenomenon is likely driven by the high surface energy inherent in nanomaterials. The statistical analysis of the particle size distribution confirms a range between 33 nm and 64 nm, successfully validating the nanometric nature of the product. Such features, including high surface area and dense grain packing, are pivotal for enhancing the physicochemical and catalytic performance of the particles. Interestingly, while the FE-SEM showed larger clusters, the mean particle size was calculated at 12 nm (Fig. 2).
The crystalline integrity of the samples was further examined via X-ray diffraction (XRD). The resulting pattern (Fig. 3) displayed distinct diffraction peaks, with a notably sharp peak attributed to the crystalline phase of the material. The presence of these well-defined peaks confirms the transition from an amorphous precursor to a highly crystalline nanostructure, consistent with the standard lattice parameters for such formations.

Optical Properties (UV-Vis Spectroscopy)
The optical transformation during the synthesis was monitored using UV-Visible spectroscopy (Fig. 4). The U. pilulifera extract (PE) exhibited a characteristic absorption maximum at 280 nm. Upon the formation of nanoparticles, a distinct red shift was observed with the emergence of a new secondary peak at 450 nm. This dual-peak profile retaining the extract’s signature at 280 nm while developing a new band in the 400–450 nm range serves as definitive evidence for the successful bio-reduction and formation of the nanoparticles of (ZnO NPs).

FTIR Analysis and Functional Groups Involvement
To identify the biochemical constituents responsible for the reduction and stabilization of the ZnO NPs, Fourier Transform Infrared (FTIR) spectra were recorded (4000–400 cm-¹). The crude plant extract displayed vibrational bands at ~3400 cm-¹ (O-H), ~2920 cm-¹ (C-H), ~1700 cm-¹ (C=O), and ~1050 cm-¹ (C-O), indicating a richness in phenols, flavonoids, and proteins. Post-synthesis, these peaks underwent significant intensity reductions and positional shifts, suggesting that these functional groups actively participated in the coordination and reduction of zinc ions.
A definitive “fingerprint” of ZnO formation was identified in the low-frequency region (450–520 cm-¹). This intense absorption band corresponds to the Zn–O stretching vibrations, confirming the development of a wurtzite-phase crystalline structure. The absence of this specific band in the raw extract spectrum further validates the chemical conversion of precursors into nano-oxide forms.

Proposed Mechanism: Biogenic Reduction and Capping
The spectral data supports a dual-action role for the U. pilulifera extract as both a reducing and capping agent. The modification of hydroxyl (O-H) and carbonyl (C=O) signatures implies their direct role in the electron transfer process required to reduce Zn2+ ions. Moreover, the retention of organic residues (such as amide and C-N bands) on the nanoparticle surface indicates that bioactive macromolecules (proteins and polysaccharides) formed a protective “capping” layer. This naturally occurring coating provides steric stabilization, which effectively inhibits excessive agglomeration and maintains the long-term structural stability of the nanoparticles.

Cytotoxicity of ZnO NPs Against U87 Cells
The cytotoxic activity of ZnO NPs was evaluated using the MTT assay. Results demonstrated a dose-dependent inhibition of U87 cell proliferation. At lower concentrations (0.00125–0.005 ppm), moderate reductions in viability were observed. At the highest concentration (0.01 ppm), cell viability significantly decreased, with survival reduced to \~88.35%. Microscopic examination revealed clear apoptotic changes, including cell shrinkage, rounding, membrane blebbing, and detachment.

Morphological Changes in U87 Cells
The results showed that ZnO NPs prepared using U. pilulifera(L.) significantly affected U87 brain cancer cells in a concentration- and time-dependent manner (after 48 hours of exposure) at a concentration of (0.00125)ppm it led to a reduction in the size of cancer cells and the appearance of some clusters, which indicates the onset of a cytotoxic effect, possibly by inducing intracellular stress, at a concentration of (0.0025) ppm clear signs of cell killing appeared, which indicates that the toxic effect has reached a level at which actual cell death occurs, The highest kill was at (0.01)ppm a mean 311 error rate 2.16025 with significant differences 1.08012. voids appeared in the place of dead cells, which indicates a massive destruction of cancer cells in this area, as can be seen in Tables 4 and 5 and Fig. 6.
The results of this study indicate that nettle extract (U. pilulifera L.) has clear antitumor activity, particularly against glioma brain cancer cells. The data showed a significant decrease in cancer cell viability in a concentration- and time-dependent manner, consistent with previous research on the cytotoxic effects of plant compounds extracted from nettle [26]. This effect is likely related to estrogen receptor pathways, as other studies have shown [27]. These results are also consistent with the findings of Kriegl et al. [28], who indicated that the bioactive components of nettle can stimulate apoptosis signaling pathways and inhibit cancer cell division by affecting chromatin receptors, leading to accelerated cell death. Previous studies, consistent with the results of the current research, have shown that the increase in apoptosis rate is directly proportional to both time and dose. This suggests that nettle extract not only inhibits cell division but also effectively activates apoptosis mechanisms. The results also showed that the extract’s toxic effect on normal human fibroblasts was limited (<10%), indicating high selectivity toward cancer cells, a key factor in modern therapeutic applications. This selective toxicity is consistent with other reports that have confirmed the safety of nettle extracts on healthy cells [29]. Overall, the study concluded that the green synthesis of ZnO NPs susing nettle extract contributes to inducing apoptosis in cancer cells with very little effect on normal cells. The results recommend expanding future studies to explore the effectiveness of these particles against other types of cancerous tumors, to evaluate their potential as a promising therapeutic agent in the field of nanomedicine.

Limitations
Despite the promising anticancer activity of ZnO NPs synthesized using nettle, this study has several limitations. First, all in vitro experiments were performed on a single cancer cell lineage (U87), limiting the extrapolation of results to in vivo conditions. The molecular mechanisms of cytotoxicity were not fully elucidated, as no apoptosis markers, reactive oxygen species (ROS) levels, or gene expression analyses were performed. Furthermore, important nanoparticle properties, such as zeta potential, DLS, and long-term stability, were not assessed. The study also did not compare the nanoparticles to standard chemotherapeutic agents. The phytochemicals responsible for the observed activity were not quantified using advanced analytical techniques. Therefore, further in vivo studies, mechanistic investigations, and broader cytotoxicity assays are needed to verify the therapeutic potential of ZnO NPs.

CONCLUSION
The current study demonstrated anticancer activity against tested cancer cells. Cell proliferation was inhibited and apoptosis was accelerated. Microscope images showed that the effect targeted cancer cells but not healthy cells. The results showed that the lethal effect of the nanomaterials and extract increased with increasing concentration and time. These findings suggest that nettle extract could be a promising natural anticancer agent. Future research should focus on isolating the active phytochemicals, elucidating the molecular mechanisms underlying its anticancer effects, and conducting in vivo studies to verify its efficacy and safety in potential therapeutic applications.

ACKNOWLEDGMENTS
Many thanks and gratitude to the University of Kufa, College of Science, Department of Ecology for the scientific support, laboratories and consultations they provided.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.