A549 cells, which are derived from human adenocarcinomic alveolar basal epithelial cells, have been crucial in advancing the study of lung cancer biology, particularly in the areas of tumor progression, metastasis, and therapeutic response. These cells are frequently used because of their consistent and reproducible experimental results, as well as their simplicity of cultivation in laboratory setting (Giannopoulos-Dimitriou et al., 2024). The A549 cell line used in the present study was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. Throughout the duration of the study, the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12. The culture media was supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (consisting of penicillin and streptomycin) to promote cellular proliferation and protect against microbial pathogens. The A549 cells were cultivated in a CO₂ incubator maintained at a temperature of 37°C and immersed in a 5% CO₂ environment. The medium underwent refreshment at intervals of 48 h or earlier in the event of a yellow discoloration, which signifies a change in pH or depletion of nutrients. Once the cell density reached around 80% confluence, which signifies a culture that is healthy and actively growing, the cells were ready for passaging. To allow their transfer to other culture vessels for further growth, the cells were detachable using a 0.25% trypsin solution with 0.02% ethylenediaminetetraacetic acid (EDTA).

A dose-response study was conducted to evaluate the cytotoxicity of Au-NPs on A549 lung cancer cells. The study employed doses ranging from 1 to 50 μg/mL and subsequently conducted an MTT assay (Mosmann, 1983). To assess the influence of Au-NPs on mitochondrial integrity, the production of formazan crystals was observed. These crystals are formed as a consequence of the reduction of MTT by mitochondrial succinate dehydrogenase, which is commonly released by hepatic cells. In this experimental study, cells were dissociated using trypsin and subsequently measured using a cell counter. The aim was to allocate roughly 10⁵ cells per well in 96-well plates. The lung cancer cells were exposed to various concentrations of Au-NPs, ranging from 0 to 50 μg/mL, and then placed in a 37°C environment with a 5% CO₂ atmosphere for a duration of 24 h. The determination of cell survival rates was conducted using the MTT test, which allowed for the identification of the median cytotoxic concentration (IC50) of Au-NPs for further inquiry.

The investigation into the anti-cancer properties of biosynthesized Au-NPs entailed an analysis of the alterations in cell shape following exposure to Au-NPs. The TUN staining was used to accomplish this. Each well of 6-well plates was seeded with approximately 200,000 cells, which were subsequently treated to doses of 15 and 20 μg/mL of Au-NPs. After being incubated in a 5% CO₂ atmosphere for 24 h at 37°C, the cells were fixed for 15 min using a solution containing 2.5% glutaraldehyde and 0.1% Triton X-100. Following fixation, the cells were subjected to DAPI staining for 5 min at 37°C. This process aimed to emphasise the nuclei, making it easier to detect any alterations that may indicate apoptosis or necrosis. The cells were rinsed with ice-cold PBS after DAPI staining in order to eliminate any remaining stain. The NIKON Eclipse 80i fluorescent microscope was utilised to conduct observations on the labelled lung cancer cells, facilitating a comprehensive visualisation of the cellular reactions to Au-NPs treatment.

Assays were conducted utilising kits obtained from China to assess the effects of Au-NP on caspases, which are crucial proteins that trigger apoptosis. The experiments employed the p-NA substrate, which emits light when broken down by caspases. The DEVD sequence is recognised and cleaved by caspase 3, whereas the LEHD sequence present in the p-NA substrate is targeted and cleaved by caspase 9. This process leads to the generation of light, which was measured at a wavelength of 400 nm. This technique enables the quantification of caspase activity, therefore evaluating the inducer of cell death caused by Au-NPs.

The fluorescent probe known as 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) is a derivative of fluorescein that exhibits cellular permeability. It functions as a highly sensitive detector for ROS. The conversion of H₂DCFDA from a non-fluorescent state to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) occurs upon its contact with ROS. This transformation enables the quantification of H₂DCFDA at a wavelength of 530 nm (Klastersky et al., 1989). The conversion of ROS is of utmost importance in the investigation of the impact of anti-cancer drugs on the apoptotic process. The presence of Au-NPs has been demonstrated to increase levels of ROS, which can be accurately measured using the H2DCFDA assay kit. The experiment involved cultivating A549 cells in 6-well plates and subjecting them to Au-NPs at dosages of 15 and 20 μg/mL for a period of 24 h. After the treatment, the cells were incubated at 37°C for 30 min with a concentration of 80 mM of the H₂DCFDA dye. Following that, the fluorescence intensity at 530 nm was measured using an ELISA reader. The results for each sample were corrected according to the protein content in order to maintain accuracy.

The influence of Au-NPs therapy on mitochondrial membrane integrity in A549 cells was disclosed using fluorescence microscopy, with rhodamine 123 serving as a selective marker (Rajivgandhi et al., 2018b). The observation of cyt-c efflux from the mitochondria into the cytosol upon exposure to the IC50 concentration of Au-NPs is a significant milestone in the process of apoptosis, indicating the potential anti-cancer properties of Au-NPs. This phase highlights the initial interaction between Au-NPs and A549 cells, resulting in the disruption of mitochondria. The cell cultures that had reached maturity on coverslips were subjected to the IC50 concentration of Au-NPs and thereafter incubated for 24 h at 37°C. After the incubation period, the cells were subjected to centrifugation and subsequently subjected to trypsinization. Following the process of trypsinization, a washing step was conducted using 1x PBS. The cells on coverslips were treated with Rhodamine 123 dye at a dosage of 5 μg/mL for a duration of 1 h. Following this time frame, any excess dye was eliminated using Whatman No.1 filter paper and 1x PBS. In order to differentiate between the modified structures of impaired and undamaged mitochondrial membranes in the A549 cell line, a fluorescent microscope was employed to investigate both the treated and control cell groups.

The synthesis of Au-NPs was confirmed using UV-Vis spectroscopy. The absorbance spectra exhibited a range of values between 350 and 700 nm, with the most prominent peak observed at 540 nm as depicted in Figure 1. It is found that the Au-NPs formation was completed within 15 min at room temperature. However, when the reaction temperature was maintained at 100°C, the reaction was completed within 5 min (Supplementary Figure S1). This peak is a direct indication of the SPR effect, confirming the formation of Au-NPs. This peak is within the expected range for gold nanoparticles, indicating successful synthesis. The size and shape of the nanoparticles were exhibited depending upon the SPR property of the metal (Fayaz et al., 2010).

The biomolecules included in the Au-NPs samples were identified and their ability to create a capping layer was assessed using FTIR spectroscopy (Figure 2). Prominent absorption peaks were seen in the spectrum analysis, notably at 1,528, 1,747, 2,335, and 2,933 cm⁻¹, within the range of 1,000–4,000 cm⁻¹. The observed peaks are caused by the interaction and reduction of gold ions due to the presence of compounds in M. oleifera. The presence of hydroxyl groups in polyphenolic compounds from M. oleifera is indicated by the absorption peak at 2,933 cm⁻¹. The peak observed was due to the decrease of gold content which may be due to the presence of polyphenols that binds the gold with carbonyl group and cap the Au-NPs hence preventing from combination of nanoparticles (Ali et al., 2011). The functional components of M. oleifera could have bound to AuCl₄ and decreased it to the formation of Au-NPs. Additionally, the inclusion of ketones, aldehydes, and esters within the nanoparticle structure is suggested by the peak at 2,335 cm⁻¹. Polyphenols, including quercetin and gallic acid, have been shown to inhibit cancer cell growth and induce apoptosis in lung cancer cells (Mumtaz et al., 2021). Flavonoids like kaempferol and catechins have demonstrated anti-inflammatory and anti-proliferative effects on lung cancer cells. They can modulate signalling pathways involved in cancer cell survival and proliferation (Wu et al., 2021). Phenolic compounds can enhance the immune response against cancer cells by promoting the activity of immune cells such as T-cells and macrophages. They also help in reducing tumor-associated inflammation (Wang et al., 2023). Isothiocyanates found in M. oleifera, have been shown to inhibit cancer cell migration and invasion, thereby preventing metastasis (Wu et al., 2021).

TEM was used to assess the shape and structural characteristics of Au-NPs obtained from the leaf extract of M. oleifera. Figures 3A, B presents TEM images illustrating Au-NPs with diameters varying between 20 and 40 nm, most of them displaying a spherical shape. The spherical morphology of Au-NPs observed in the TEM results provides several advantages for biomedical applications, including enhanced stability, dispersibility, cellular uptake, and functionalization capabilities (Georgeous et al., 2024). These properties make spherical Au-NPs particularly suitable for targeted drug delivery and cancer therapy.

The production and stability of Au-NPs by plant-derived phytochemicals were further confirmed using EDX examination. The EDX profile of Au-NPs as depicted in Figure 4 exhibited strong Au signals corresponds to elemental Au-NPs and additional peaks corresponding to carbon, oxygen were also observed. These peaks may be attributed to biomolecules present in the extract (Moodley et al., 2018). The elemental percentages were shown in Table 1.

The study further confirmed the crystallinity of the Au-NPs using (SAED) analysis, as shown in Figure 5. The pattern seen in the SAED analysis exhibited prominent, concentric rings, which provide as evidence for the crystalline arrangement of the Au-NPs. The diffraction patterns observed in these rings are a result of the different lattice planes present in the crystalline Au-NPs. SAED pattern is attributed to the (111), (200), (220) and (311) planes of face centered cubic compatible with the XRD.

The crystalline structure of the Au-NPs was definitively determined using XRD research. The XRD spectrum, as illustrated in Figure 6, exhibited four distinct diffraction peaks at angles of 38.12°, 44.87°, 64.77°, and 77.23°. The peaks are indicative of the lattice plane indices (111), (200), (220), and (311), which are indicative of the face-centered cubic (FCC) structure of gold (Au). The measured lattice parameter of a = 4.08A° exhibits a strong agreement with the established values for FCC gold, as supported by the reference data obtained from JCPDS Card No-04-0784.

DLS analysis was used to determine the overall size and thickness of the capping layer on the nanoparticles. The mean size of the Au-NP was determined to be around 30 nm using this methodology, as depicted in Figure 7. The polydispersity index, which quantifies the range of particle sizes, was found to be approximately 0.321%. The results of the characteristic analysis were compared with the results of the previous studies, showed in Table 2 (Guliani et al., 2021; Khan et al., 2021; Gopinath et al., 2014).

The cytotoxic effects of different doses of Au-NPs on the lung cancer A549 cell line are depicted in Figure 8, which was evaluated using the MTT assay. Determining the IC50 value helps in identifying the optimal dose range for a drug candidate. Using IC50 values helps in finding the lowest effective dose that minimizes harmful effects on normal cells (IC50, 2025; Robles-Bañuelos et al., 2024). IC50 value was obtained at dose of 50 μg/mL which inhibited the growth of 50% A549; hence, this dosage was selected for the analysis of anticancer effect of Au-NPs. In order to further investigate the impact of Au-NPs on cell survival, a concentration of 50 μg/mL was used for following studies, as well as a reduced concentration of 25 μg/mL. The high concentration of bioactive metabolites in M. oleifera leaf extracts, which substantially contribute to the induction of cytotoxicity, can be attributed to the cytotoxic effect of Au-NPs (Gismondi et al., 2015). Furthermore, the interaction between gold atoms and intracellular components plays a crucial role. Gold ions disrupt cellular function and contribute to the observed cytotoxic effects by interfering with intracellular proteins, as well as phosphate and nitrogen groups in DNA (Blagoi et al., 1991). In the previous studies phenolic compounds of M. oleifera leaf extracts were tested for their anti-cancer activity against Hela cancer cells in which n-hexane fraction showed a 50% reduction in Hela cancer cell viability at 416 μg/mL compared to control (Mumtaz et al., 2021). Some previous studies reported an IC50 value of 11.13 μg/mL for AS1411 aptamer-conjugated, green-synthesized AU-NPs against MCF-7 breast cancer cells (Helal et al., 2024) and 10 μg/mL for Au-NPs combined with metformin against MCF-7 and A549 cells (Yeşildağ et al., 2024).

The study examined the ability of Au-NPs to induce apoptosis in the lung cancer A549 cell line using DAPI labelling. The DAPI staining findings are depicted in Figures 9A–C, illustrating the comparison between control cells and cells treated with Au-NPs. The nuclei in the control samples were found to be undamaged, whereas the application of Au-NPs at doses of 25 μg/mL and 50 μg/mL led to a decrease in the cell viability. Au-NPs can generate ROS which are oxygen-containing molecules with a high degree of chemical reactivity. Oxidative stress can be induced by the accumulation of ROS, which can result in the destruction of critical cellular components, including DNA, proteins, and lipids (Foglietta et al., 2023). The release of pro-apoptotic factors, such as cytochrome c, into the cytoplasm can be a consequence of elevated ROS levels, which can impair mitochondrial function. The activation of caspases, a class of proteases that mediate apoptosis by cleaving specific cellular targets, is initiated by this event (Lee et al., 2022). Furthermore, ROS have the potential to cause DNA damage, which in turn activates the p53 pathway, thereby contributing to apoptosis and cell cycle arrest (Foglietta et al., 2023; Lee et al., 2022). Elevated ROS levels, caspase activation, DNA and protein degradation, and alterations in the cell membrane are among the primary biochemical changes associated with apoptosis (Kumar et al., 2010). An increase in intracellular ROS levels and activities of caspases 3 and 9 has been observed in lung cancer cells treated with Au-NPs, confirming the induction of apoptosis in the A549 lung carcinoma cell line. This apoptotic process was further verified by DAPI staining. Previous studies revealed that Au-NPs induced apoptosis and inhibited cancer cell proliferation (Siddique et al., 2022).

In the process of apoptosis, caspases play a vital role, wherein Caspase 3 acts as an initiating caspase and Caspase 9 acts as an executing caspase. The caspase activity in untreated control cells and lung cancer A549 cells subjected to different dosages of Au-NPs is depicted in Figures 10A, B. A549 cells exposed to concentrations of Au-NP at 25 and 50 μg/mL exhibited a notable elevation in both initiating and executing caspase levels. Specifically, cells treated with 25 μg/mL of Au-NPs shown a nearly 1.5-fold increase in caspase activity. The data presented provide evidence of the significant apoptotic impact of Au-NPs on lung cancer cells through the augmentation of caspase activation. The anticancer activity of Au-NPs is strongly supported by the elevated levels of caspase 3 in A549 cells. An increase in the protein expression of Beclin 1, a mammalian orthologue of yeast Atg6, further corroborated this effect. Beclin 1 is essential for the regulation of apoptosis, and its activity is inhibited by Bcl2, which binds to the BH3 domain of Beclin 1 to suppress its function (Kang et al., 2011). Treatment with AuNPs led to a decrease in Bcl2 protein expression, which likely facilitated the elevated levels of Beclin 1, thereby inducing apoptosis in the A549 lung carcinoma cell line.

This study illustrates the comparison of ROS levels in A549 lung cancer cells that were treated with Au-NPs and those that were not treated (control). The administration of Au-NPs at concentrations of 25 μg/mL and 50 μg/mL led to a 50% and 100% rise in ROS generation, indicating that the biosynthesized Au-NPs induces apoptosis (Figure 11). The observed rise in ROS levels is associated with an elevated occurrence of apoptotic cells as identified by DAPI staining. Activators of the intrinsic apoptotic pathway are recognised for their ability to elevate intracellular ROS and cytosolic calcium levels (Kang et al., 2011). The levels of ROS in A549 cells were substantially increased by the treatment with Au-NPs in this study. The protein expression of intrinsic apoptotic signalling molecules was analysed in both control and Au-NPs treated cells to further investigate the mechanism. A significant change was observed in the Bcl2 family of proteins, which encompasses both pro-apoptotic and anti-apoptotic members. The pro-apoptotic proteins Bid and Bax were upregulated, while the anti-apoptotic protein Bcl2 was downregulated (Karp, 2008). It is well-established that Au-NPs catalyse the production of ROS through electron transfer reactions, which leads to the formation of reactive species including superoxide anions, hydrogen peroxide, and hydroxyl radicals. AuNPs can also discharge metal ions that contribute to the generation of ROS. The cellular antioxidant defences can be overpowered by the elevated ROS levels, which can result in oxidative stress and cellular injury (Reed, 1997). The importance of ROS in the targeting of cancer cells has been emphasised in previous research. For example, Naveen kumar et al. (2018b) demonstrated that AgNPs significantly induced oxidative stress responses in A549 cells, thereby establishing ROS as a critical factor in cancer inhibition. In the same vein, Bhakya et al. (2016) reported that the integrity of the mitochondrial membrane was disrupted in A549 cells as a result of the oxidative stress induced by higher concentrations of AgNPs. This resulted in the downregulation of anti-apoptotic genes, which in turn triggered programmed cell death through mitochondrial dysfunction and intracellular leakage.

After 24 h of incubation period, the use of fluorescence microscopy demonstrated the presence of cyt-c leakage from the mitochondrial membrane subsequent to the application of Rhodamine 123 labeling, as depicted in Supplementary Figures S2A, B. Rhodamine 123 has been acknowledged for its effectiveness in detecting changes in the membrane structure of malignant cells through the disruption of the intrinsic route of the cancer cell cycle. This dye exhibits specific affinity for dysfunctional mitochondrial membranes, hence identifying the impacted cells. Cellular damage results in the development of apoptotic and necrotic cells, which exhibit a noticeable change in fluorescence from orange to green. This change emphasizes the important observation of mitochondrial membrane depolarization caused by gene failure. The activation of inhibitory genes, including as BCL-2 and caspases, is initiated by damage to the mitochondrial membrane, resulting in a reduction in gene production that facilitate the proliferation of cancer cells. Introduction of Rhodamine 123 into the mitochondrial membrane leads to the removal of internal leakage materials and a color transition from red to green, which signifies apoptosis (Momtazi-borojeni et al., 2013). The findings of this investigation provide additional evidence of the inhibition of A549 cell proliferation at the IC50 concentration of Au-NPs. The treated samples exhibited a significant rise in apoptotic and necrotic cells when compared to the untreated control group, as depicted in Supplementary Figures S2A, B. This finding suggests that the apoptotic cell receptor cascade is activated in A549 cells, leading to a notable disruption in the course of the cell cycle. This disruption is attributed to an increased activation of gene response, which signifies the onset of apoptosis. The results presented in Supplementary Figure S2B illustrate the presence of rough, loosely connected apoptotic cells exhibiting a clear necrotic feature, while Supplementary Figure S2A showcases smooth, densely packed cell colonies. These findings align with the results reported by Rajivgandhi et al. (2019b) in which observed reduction in red fluorescence intensity indicates a notable decline in the integrity of the mitochondrial membrane. The efficacy of cancer cell therapy procedures is attributed to the loss and functional degradation of the mitochondrial membrane, which results in heightened cell mortality. The findings of our study are consistent with the research conducted by Du et al. (2017), which highlights the significance of mitochondria in initiating cellular signaling pathways, promoting cell differentiation, inducing apoptosis, and regulating the course of the cell cycle. In Table 3 we have provided the comparison table of previous studies (Liu et al., 2013; Niloy et al., 2021; Ramalingam et al., 2017; Tanino et al., 2020; Rani et al., 2022; Hongal et al., 2024; Dehnoee et al., 2024; Kalaiarasi et al., 2018).

While the present findings provide a promising foundation for lung cancer treatment, further studies are necessary to evaluate the in vivo safety, biocompatibility, and potential toxicity of these nanoparticles. Addressing these concerns is critical for the translation of these results into clinical applications.