Green chemistry-based synthesis of gold nanoparticles using Nicotiana plumbaginifolia and their anticancer properties against cervical cancer

2.1 Materials

Gold chloride trihydrate (III) (HAuCl₄·3H₂O, CAS No.:16961-25-4), bovine serum albumin (BSA CAS No.: 9048-46-8), deoxyribonucleic acid sodium salt from calf thymus (DNA, CAS No.: 73049-39-5), 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA, CAS No.: 4091-99-0), Z-DEVD-FMK (caspase-3 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, CAS No.: 298-93-1] were purchased from Sigma Co. (MO, USA). For DNA and BSA binding assays, 0.01  M tris-HCl buffer solution was used for the preparation of the working solutions. The primers for the quantitative real-time PCR (qPCR) study were obtained from Genscript (China). Dulbecco’s modified Eagle’s medium (DMEM), N-Acetyl-L-cystein (NAC), and fetal bovine serum (FBS) were purchased from Gibco (USA). The medium 199 (M199) was purchased form from HyClone (Thermo Fisher Scientific, Waltham, MA, USA). All other materials were of analytical grade and aqueous solutions were prepared using double-distilled water. The number of AuNPs per liter, the molar absorbance ɛ, and the concentration of AuNPs were determined in terms of the equations previously reported by Tournebize et al. (2011). The working solution of the AuNPs (1 mM) was prepared by dissolving in methanol. The final concentration of methanol in different assays did not exceed 1 %.

2.2 The extraction process of Nicotiana plumbaginifolia leaf

Nicotiana plumbaginifolia leaves taken from the native habitats were washed properly with distilled water and chopped. For the preparation of the aqueous extract of the plant leaves, 10 g of leaves were crushed using a mortar pestle and added with 100 mL distilled water, heated at 70 °C for 10 min, cooled, and filtered using Whatman No. 1 filter paper. Finally, the sample was stored in the refrigerator.

2.3 Green synthesis of AuNPs

10 ml of plant extract was added to 1 mM solution of HAuCl₄·3H₂O with a final volume of 100 ml. The sample was then stirred for 50 min (100 RPM) at room temperature and centrifuged at 8000 rpm for 20 min. Afterward, the pellet was collected, washed with ethanol and distilled water and dried at 60 °C

2.4 Characterization of greenly synthesized AuNPs

The synthesis of AuNPs was assessed by analyzing the UV absorption range using a UV–vis spectrophotometer (UV–Vis, PerkinElmer – Lambda 750) at the wavelength of 300–800 nm. FTIR analysis was also performed to evaluate the chemical bonds and functional groups. The dried powder of the AuNPs synthesized was mixed with KBr and analyzed by FTIR (Spectrum 65, Perkin Elmer). Transmission electron microscopy (TEM) was used to analyze the morphology and size of the AuNPs. After dissolving the NPs in ethanol, 10 µl of the AuNPs was dropped on the copper-coated carbon grades and left to dry at room temperature. Then, the TEM images were taken on a TEM machine (TECNAI-G-20). The X-ray diffraction (XRD) method was employed to reveal the metal nature of the synthesized AuNPs on an XRD apparatus (Panalytical, X’ pert PRO-MPD, Netherlands) using CuKα radiation (λ = 0.15405 nm). Dynamic light scattering (DLS) study was done to measure the hydrodynamic diameter and zeta-potential of AuNPs using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK), equipped with a 633 nm He/Ne laser, scattered data angle of 173°. The AuNPs were filtered (0.2 µm, Milli-Q water) and diluted (1:10 for DLS and 1:1000 for zeta potential).

2.5 Albumin and DNA binding measurements

BSA and DNA binding of greenly synthesized AuNPs were evaluated based on the method reported previously (Duan et al., 2024). Briefly, macromolecule solutions were mixed with different dispread solutions of AuNPs ranging from 0.01 to 0.1 % with a 1:1 ratio. After 30 min, the samples were centrifuged, and the concentration of protein and DNA were determined spectroscopically at 280 and 260 nm, respectively.

2.6 Hemolysis assay

The hemolysis assay for greenly synthesized AuNPs was done based on the method reported previously (Behera and Awasthi, 2021). Blood samples taken from the blood bank were drawn into K2-EDTA-coated Vacutainer tubes. Briefly, isolated red blood cells (RBCs) were treated with different concentrations of AuNPs ranging from 0.01 to 20 µM at 37 °C for 1 h followed by centrifugation at 300 rpm for 5 min. Then, 100 µl of supernatant was removed and the optical density of samples was read at 590 nm using a microplate reader (Epoch, BioTek Instruments, USA). Positive control and negative control were considered as 100 μl of 1 % SDS and 100 μl of PBS, respectively, +50 μl of cells. The hemolysis (%) was calculated based on the equation reported previously (Singh et al., 2020).

2.7 Cell culture

The human cervical cancer HeLa cells and Human umbilical vein endothelial cells (HUVECs) were obtained from the Tumor Hospital of the Chinese Academy of Medical Sciences. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10 % (v/v) fetal calf serum, 1 % penicillin–streptomycin-glutamine (Invitrogen) in a humidified incubator containing 5 % CO₂ at 37 °C. HUVECs as the normal cells were cultured in M199 containing 10 % FBS, 1 ng/ml basic fibroblast growth factor (bFGF; Invitrogen), and 1 % penicillin–streptomycin-glutamine (Invitrogen) at 37 °C with a humidified atmosphere of 5 % CO₂ in air.

2.8 MTT assay

The viability of the cells was assessed by MTT assay. The cells (5x10³ cells/well) seeded in 96-well plates were treated with AuNPs at different final concentrations of 0.01–20 μM for 24 h. The maximal concentrations of the methanol used in all cellular experiments were 3.0 % (v/v). After incubation, the cells were prepared for MTT assay. 20 μL of MTT solution with a concentration of 5 mg/mL was added to each well and incubated at 37 °C for 4 h. The supernatant was then removed and formazan precipitates were added by 150 μL of dimethyl sulfoxide (DMSO). After 15 min, the absorbance of samples was read at 490 nm on a microplate reader (Epoch, BioTek Instruments, USA).

2.9 ELISA assay

The quantitative determination of cytochrome C (Cyt. C), caspase-3, and caspase-9 in cell lysate of cells treated with 1.5 µM greenly synthesized AuNPs for 24 h was done by ELISA assay using relevant ELISA Kit (Cusabio Biotech Co., LTD, Wuhan, China), according to the instructions.

2.10 Detection of mitochondrial membrane potential (MMP)

For the detection of MMP collapse, cells treated with 1.5 µM greenly synthesized AuNPs for 24 h were incubated with Rh123, 5 μg/ml, for 45 min in the dark at 37 °C. Then, the cells were harvested, suspended in PBS, and the fluorescence intensity changes were determined using a fluorescent microplate reader (XFLUOR4, GENios) with an excitation peak at 508 nm and an emission peak at 528 nm.

2.11 Reactive oxygen species (ROS)-generation assay

Intracellular ROS generation was determined by the DCFH-DA probe. Cells plated in 6-well plates and attached overnight were treated with 1.5 µM greenly synthesized AuNPs for 24 h. The cells were then harvested, washed twice with PBS, resuspended, and incubated with 10 μM DCFH-DA for 15 min at 37 °C. The cells were then washed with PBS, resuspended, and cell fluorescence was read on a fluorescent microplate reader (XFLUOR4, GENios) at excitation and emission wavelengths of 480 nm and 530 nm, respectively. For ROS-related studies, the cells were pretreated with NAC (10 μM) for 2 h.

2.12 RNA extraction and RT-qPCR

After treating the cells with 1.5 µM greenly synthesized AuNPs for 24 h, total RNA was extracted by the standard protocol using TRIzol Reagent (TaKaRa, China) followed by synthesis of the first-strand cDNA using PrimeScript™ RT reagent Kit (TaKaRa, China) based on the manufacturer's protocols. The qPCR reaction was done on a Real-Time machine (Roche, Basel, Switzerland) with a SYBR® Green Premier Pro Taq HS qPCR Kit. All primers used were designed based on the previous study (Xu et al., 2021). The expression of the genes of interest was calculated using the 2^−ΔΔCT method.

2.13 Caspase-3 and caspase-9 activity assays

The cells treated with 1.5 µM greenly synthesized AuNPs for 24 h, were harvested for caspase analysis using Caspase-3 and Caspase-9 Colorimetric Assay Kits (R&D System Inc.) in accordance with the manufacturer's instruction. Totally, after cell lysis and centrifugation, the samples were incubated with specific caspase-3 substrate (DEVD-pNA) or caspase-9 substrate (LEHD-pNA) for 1 h at 37 °C and the optical densities of samples were read at 405 nm.

2.14 Determination of effect of caspase inhibitors by MTT and flow cytometry assays

To determine the apoptosis signaling pathway induced by AuNPs, the cells were pretreated with 20 µM each of Z-DEVD-FMK (caspase-3 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) for 2 h. Then, cells were incubated with 1.5 µM greenly synthesized AuNPs for 24 h and cell survival was assessed using MTT assay as described above. Also, flow cytometry analysis for the detection of apoptosis was done using the staining of cells with Annexin-V/FITC. For this reason, the cells were washed, suspended in the binding buffer as instructed by the manufacturer's protocol (Sigma, USA), incubated with 1.5 μl of Annexin-V/FITC for 15 min in dark, diluted with PBS, and finally analyzed using FACScan cytometer (FACSCalibur; BectonDickinson, San Jose, CA).

2.15 Statistical analysis

Statistical analysis data were expressed as the means ± standard deviation of three experiments. The one-way analysis of variance assay was done using SPSS software. Values of p < 0.05 were reported statistically significant.

3.1 Characterization of greenly synthesized AuNPs

Fig. 1 shows the characterization of greenly synthesized AuNPs using Nicotiana plumbaginifolia leaf extract by different techniques. The different phytochemicals present in the leaf extract such as alkaloids, polyphenols, and flavonoids serve as reducing as well as capping agents during the synthesis of NPs (Duan et al., 2024, Yadav et al., 2024). The interaction of phytochemicals with metal ions resulted in a color change of the mixture, revealing the fabrication of the NPs (Yadav et al., 2024). Following the combination of gold salt and leaf extract under different reaction conditions, the salt ions not only are reduced upon the interaction with phytochemicals but also covered with different functional groups (Yadav et al., 2024).

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Fig. 1

Cauterization of synthesized AuNPs through green chemistry method. (A) TEM, (B) XRD, (C) UV–Vis, (D) FTIR, (E) DLS, (F) Zeta potential measurements.

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Fig. 1A exhibits the morphology and size of synthesized AuNPs determined by TEM imaging. It reveals that AuNPs mostly possess spherical-shaped morphology with subtle aggregation. The nanosphere had an average particle size of 18 nm (Fig. 1A). Also, it was seen that there is almost a homogenous distribution of AuNPs with similar geometrical shapes and sizes. Overall, the outcome of TEM of AuNP samples confirmed the successful reduction of Au salt and subsequent NP development. Duan et al. showed that few spherical iron oxide NPs were synthesized using Nicotiana plumbaginifolia leaf extract and the formed NPs had a size of around 15 nm (Duan et al., 2024).

It is also important to investigate the crystal structure of the AuNPs developed and this can be addressed by analyzing the XRD pattern of the NPs. The XRD peaks at 38.00°, 44.10°, 63.75° and 77.10° are indexed to the “(1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of FCC (face-centered cubic) structure of metallic gold (JCPDS no. 04-0784)” (Suman et al., 2014), indicating that the biosynthesized AuNPs are developed based on pure crystalline Au (Fig. 1B). Also, it was observed that the peak at (1 1 1) is the most intense peak among other planes, revealing that this plane is the dominant orientation and the biosynthesized AuNPs are crystalline (Suman et al., 2014; Patil et al., 2023).

UV–Vis spectroscopy is an analytical technique that is typically used to characterize the surface Plasmon resonance (SPR) properties of NPs because of the surface plasmon resonance (SPR) phenomenon (Abdelhalim et al., 2012). SPR characteristics known as a collective excitation of the electrons are heavily associated with the size and shape of NPs. Therefore, metallic NPs could display different optical absorption signals in the UV–Vis region (Abdelhalim et al., 2012; Parthiban et al., 2023). The UV–Vis spectrum detected from NP solution after the completion of the biosynthesis process showed a maximum absorption peak at about 538 nm (Fig. 1C), attributing to the SPR features of the AuNPs (Parthiban et al., 2023).

The narrow bandwidth of SPR also indicates the biosynthesized AuNPs might possess a homogenous size distribution. Actinidia deliciosa (Naraginti and Li 2017), Fagopyrum esculentum (Babu et al., 2011), Gloriosa superba (Gopinath et al., 2016) extracts have all been employed to fabricate AuNPs with similar outcomes.

Following the combination of Au salt and leaf extract under different reaction conditions, the salt ions not only reduced following the interaction with phytochemicals but also covered with different functional groups (Yadav et al., 2024) which can be determined by FTIR study.

The FTIR spectra of Nicotiana plumbaginifolia leaf extract and biosynthesized AuNPs measured in the range of 4000–400 cm⁻¹ are shown in Fig. 1D. The Nicotiana plumbaginifolia leaf extract sample shows FTIR bands at 3340, 1713, 1581, 1362, 1189 and 1048 cm⁻¹. For plant extract sample the FTIR band at 1713 cm⁻¹ could be attributed to carbonyl peak, which might be responsible for the reduction of Au ions to AuNPs (Dubey et al., 2010). FTIR bands stemming from the —OH group at ∼3340 and 1048 cm⁻¹, and 1581 cm⁻¹ are indicative of the presence of NH2 groups (Dubey et al., 2010). Comparison between FTIR spectra of Nicotiana plumbaginifolia leaf extract and biosynthesized AuNPs displays some alterations in the wavenumber and the magnitude of the bands. For biosynthesized AuNPs a strong and wide FTIR band was detected at about 3270 cm⁻¹, attributing to the O—H stretching vibration which can be derived from plant extract or absorption of water molecules by NP samples during analysis (Barnawi et al., 2022). Moreover, the typical band at about 1720 cm⁻¹ could be assigned to C⚌ O stretching of a carbonyl group found in plant extract (Rao et al., 2019; Döll et al., 2023). It should be noted that C⚌O band position was shifted to higher wavenumbers relative to plant extract, revealing the interaction and coordination of Au with an oxygen atom in C⚌O (Barnawi et al., 2022). Also, the appeared band at 1020 cm⁻¹ is derived from C—C or C—O stretching vibration (Jayaseelan et al., 2013). An observed peak at about 769 cm⁻¹ is attributed to the Au—O stretching vibration (Suriyakala et al., 2022).

The FTIR analysis verified the capping behavior of Nicotiana plumbaginifolia leaf extract which might impart high stability to the biosynthesized AuNPs.

The DLS instrument can be used to analyze the size of NPs along with the shell thickness of biochemical agents and water molecules enveloping their core surface (Sujitha and Kannan 2013). The average size of the biosynthesized AuNPs was then detected by DLS measurement. The size distribution vs. intensity graph exhibited in Fig. 1E revealed that the maximum size for colloid AuNPs was 68.69 nm. Upon comparison of particle sizes determined by TEM (Fig. 1A) and DLS (Fig. 1E) analyses, we found that the detected particle size of AuNPs by DLS was larger than that of the TEM result. This difference is derived from the inclusion of the bioorganic agents enveloping the surface of the AuNPs in DLS measurement (Sujitha and Kannan 2013). Also, van der Waals forces between NPs appearing in the solution could affect the agglomeration of NPs and their sizes accordingly (Sujitha and Kannan 2013). Also, different sample preparations for microscopic and DLS techniques could result in different size results (Sujitha and Kannan 2013).

Zeta potential value could provide us with some useful information about the surface charge, interaction forces, and stability of the synthesized NPs (Pochapski et al., 2021). The result of the zeta potential value for biosynthesized AuNPs was −47.92 mV (Fig. 1F), indicating the strong stability of the biosynthesized NPs.

3.2 Protein and DNA binding as well as hemolysis assays

Binding assays between BSA and DNA with greenly synthesized AuNPs were evaluated by UV–Vis study. Proteins and DNA exhibit distinctive absorption at 280 nm and 260 nm owing to the presence of aromatic amino acid residues and bases, respectively. Fig. 2A shows the percentage binding of BSA and DNA with AuNPs. It was detected that after incubation of different % solution of AuNPs, 0, 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 the % BSA binding and %DNA binding were 0.00 %, 6.22 %, 10.19 %, 20.51 %, 32.82 %, 50.69 %, and 58.76 % and 0.09 %, 3.11 %, 13.56 %, 19.34 %,29.14 %, 30.24 %, and 32.15 %, respectively.

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Fig. 2

Cauterization of binding of synthesized AuNPs through green chemistry method with DNA and protein as well as hemocompatibility assay. (A) DNA and BSA binding, (B) hemolysis assay.

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In general, it was detected that the interaction of AuNPs with biomolecules depends on the concentration of NPs and types of biomolecules. As expected, BSA has a more substantial binding affinity with AuNPs compared with DNA, which might be due to the presence of 20 different amino acid residues in BSA which can be involved in the establishment of different forces with AuNPs, while DNA with four types of purine and pyrimidine bases could contribute in the formation of hydrogen bonding interactions (Duan et al., 2024).

Also, the hemolysis assay was done to analyze the hemocompatibility of biosynthesized AuNPs. It was shown that after incubation of RBCs with different concentrations of AuNPs, 0.01, 0.1, 0.5, 1, 10, and 20 μM, the hemolysis values were 0.03 %, 0.38 %, 0.72 %, 0.79 %, 1.00 %, and 2.29 %, respectively (Fig. 2B). Overall, it was detected that greenly synthesized AuNPs do not significantly trigger hemolytic activity. At the highest concentration of AuNPs, 20 μM, the hemolysis percentage of 2.29 % is detected as shown in Fig. 2B, which is whitin the safe limit and in agreement with the previous studies (Dey et al., 2016; Kumar et al., 2020). The results confirmed that greenly synthesized AuNPs might possess safe therapeutic potency since it inserts minimum hemolytic activity on human RBCs.

3.3 Growth inhibitory effects of greenly synthesized AuNPs

First, the effects of AuNPs on the growth of the human cervical cancer HeLa cells and human umbilical vein endothelial cells (HUVECs) were compared. After 24 h incubation with greenly synthesized AuNPs at concentrations ranging from 0.01 to 20 μM, the MTT assay was carried out. As depicted in Fig. 3, AuNPs were much more efficient as cell proliferation inhibitors in HeLa cells compared to normal HUVEC: the IC₅₀ value of AuNPs was 1.53 μM in HeLa cells, whereas this value for HUVEC normal cells was higher than 20 μM (30.55 μM). Indeed, after incubation of HeLa cells with different concentrations of AuNPs, 0, 0.01, 0.1, 0.5, 1, 10, and 20 μM, the cell viability was reported to be 100.05 %, 91.18 %, 82.11 %, 72.88 %, 64.93 %, 53.05 %, and 45.76 %, while these amounts for normal HUVEC were 100.05 %, 98.37 %, 91.74 %, 89.31 %, 87.33 %, 74.72 %, and 63.27 %, respectively. These data indicate that AuNPs synthesized through green chemistry route from Nicotiana plumbaginifolia leaf extract are likely to be more potent antiproliferative NPs against HeLa calls than normal HUVEC (Fig. 3). These outcomes suggest that the green synthesis of AuNPs can be a potentially important factor in determining the selective antiproliferative efficacy of AuNPs. We can also conclude that the IC₅₀ values of AuNPs depended on the cell type under investigation. Collectively, these results revealed that the synthesis of AuNPs through green chemistry played a key role in mediating the selective proliferation inhibitory effect of AuNPs in cervical HeLa cancer cells. In other words, AuNPs synthesized from Nicotiana plumbaginifolia leaf extract are likely to be more potent antiproliferative nanostructures in HeLa cancer cells than normal HUVEC.

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Fig. 3

MTT assay for determination of the effects of greenly synthesized AuNPs on the growth of the human cervical cancer HeLa cells and human umbilical vein endothelial cells (HUVECs) after 24 h. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.

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3.4 Induction of apoptosis by AuNPs

Earlier reports have shown that eco-friendly AuNPs mitigated cell growth by triggering apoptosis in different types of cancer cells including cervical cancer cells (Geetha et al., 2013; Qian et al., 2019; Dickson et al., 2023). To further investigate that weather induced growth inhibition by AuNPs synthesized through green chemistry route from Nicotiana plumbaginifolia leaf extract was linked with a capability to stimulate apoptosis, an ELISA test was employed to measure the content of released Cytc. C from mitochondria to cytosol as well as the content of caspase-9 and caspase-3. Cytc. C release and caspase-9 and caspase-3 cleavage are general features of mitochondrial-associated apoptosis. In this study, then the contents of Cytc. C, caspase-9, and caspase-3 in both Hela cancer cells and normal HUVEC were assessed by ELISA assay. Therefore, the cells were exposed to 1.5 µM AuNPs for 24 h and an ELISA assay was performed. It was shown that at 24 h incubation with AuNPs a significant increase in the levels of released Cytc. C (Fig. 4A), caspase-9 (Fig. 4B) and caspase-3 (Fig. 4C) in HeLa cancer cells were observed.

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Fig. 4

The effects of 1.5 µM greenly synthesized AuNPs on the (A) Cyt. C release, (B) caspase-9 amount, (C) caspase-3 amount, (D) caspase-9 activity, (E) caspase-3 activity, (F) MMP loss in human cervical cancer HeLa cells and human umbilical vein endothelial cells (HUVECs) after 24 h. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. ^##P < 0.01, ^###P < 0.001 compared to HeLa cells.

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Also, it was found that the elevation of these markers was significantly high in HeLa cancer cells, but not in AuNP-treated HUVEC, compared with control cells. The same trend was also observed for caspase-9 (Fig. 4D) and caspase-3 activity (Fig. 4E) assays. Moreover, the extent of MMP loss, another indicator of mitochondria-mediated apoptosis, induced by AuNPs was further decreased in HeLa cancer cells compared with normal HUVEC (Fig. 4F). Indeed, MMP analysis done after 24 h of incubation of cells with 1.5 μM AuNPs indicated that synthesized AuNPs could induce MMP collapse in HeLa cancer cells, whereas exposure to AuNPs afforded only a subtle MMP loss in normal HUVECs. These data indicate that proliferation inhibition induced by greenly synthesized AuNPs after 24 h was preceded by mitochondria-mediated apoptosis.

3.5 Stimulation of ROS generation and subsequent apoptosis induced by AuNPs

Oxidative stress mediated by NPs has been reported to be associated with apoptosis in various cell lines (Enea et al., 2020; Ozcicek et al., 2021). Also, it has been shown that AuNPs-induced apoptosis is upregulated by stimulation of ROS generation in several cancer cells (Liu et al., 2013; Kondath et al., 2020). To explore whether apoptosis-inducing abilities induced by AuNPs were afforded by changes in ROS-generating possibility, DCF fluorescence intensity as a marker of intracellular ROS level was detected after treatment of cells with 1.5 µM AuNPs for 34 h. AuNPs significantly elevated DCF fluorescence intensity compared to that of negative control HeLa cancer cells, whereas AuNPs showed no such effect in normal HUVECs, thus indicating that AuNPs are more proficient ROS inducers in HeLa cells than normal cells (Fig. 5A). Moreover, pretreatment with NAC (10 µM), a well-known ROS scavenger, remarkably decreased AuNPs-induced ROS generation in HeLa cells (Fig. 5A). These data indicate that greenly synthesized AuNPs are potent ROS inducers in HeLa cancer cells. It was then sought to determine whether AuNPs-induced ROS generation contributed to the induction of apoptosis. For this reason, the expression of Bax, caspase-9, and caspase-3 mRNA (apoptotic markers) and Bcl-2 mRNA (antiapoptotic marker) in AuNPs-treated cells were explored. In fact, it has been reported that inorganic NPs-induced apoptosis is regulated by Bax/Bcl-2, caspase-9, and caspase-3 mRNA expression (Katifelis et al., 2018; Ji et al., 2019), which is indicative of mitochondrial intrinsic apoptotic pathway.

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Fig. 5

(A) The effects of 1.5 µM greenly synthesized AuNPs alone or with NAC on the ROS generation in human cervical cancer HeLa cells and human umbilical vein endothelial cells (HUVECs) after 24 h. The effects of 1.5 µM greenly synthesized AuNPs alone or with NAC on (B) Bax mRNA, (C) Bcl-2 mRNA, (D) caspase-9 mRNA, (E) caspase-3 mRNA in human cervical cancer HeLa cells after 24 h. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. ^$XXX^$P < 0.01, ^$XXX^$XXX^$P < 0.001 compared to HeLa cells.

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As shown in Fig. 5, such upregulation of Bax mRNA (Fig. 5B) and downregulation of Bcl-2 mRNA (Fig. 5C) induced by AuNPs were recovered by pretreatment of HeLa cells with NAC. Also, the caspase-9 (Fig. 5D) and caspase-3 mRNA expression (Fig. 5E) stimulated by exposure of cells to AuNPs were reduced before exposure of HeLa cells to NAC. These data suggested that AuNPs-triggered ROS generation played a key role in initiating apoptotic cell death. In agreement with these outcomes, Kowsalya et al. also reported that greenly synthesized AuNPs from leaf aqueous extract of 10 different plants with particle dimensions ranging between 2 and 26 nm stimulated mitochondria-mediated apoptosis through oxidative stress in breast cancer cells (Kowsalya et al., 2021). Furthermore, Ramalingam et al. revealed that biogenic AuNPs from an aqueous extract of Sesuvium portulacastrum L. stimulated oxidative stress and mitochondrial membrane disruption in A549 lung cancer cells (Ramalingam et al., 2016). Prasad et al. also showed that a low concentration of biosynthesized AuNPs (25 μg mL⁻¹) in combination with glutamine deprivation promoted the growth inhibition if HeLa cancer cells through oxidative stress-mediated mitochondrial damage (Prasad et al., 2022).

3.6 Apoptosis mechanism

To further explore the probable apoptosis mechanism regulated by greenly synthesized AuNPs, we used caspase-9 and caspase-3 inhibitors and then evaluated the apoptosis and cell viability rates in the HeLa cancer cell line. Indeed, to clarify the role of caspases in the AuNPs-induced apoptosis, specific caspase-9 and caspase-3 inhibitors were utilized. It was shown that the percentage of Annexin-V FITC ⁺ in AuNPs-treated cells is 4.33-fold higher than that of control cells. However, pre-treatment of cells with specific caspase-3 inhibitor Z-DEVD-FMK and specific caspase-9 inhibitor Z-LEHD-FMK could mitigate the percentage of Annexin-V FITC⁺ (%) to 2.26- and 2.53-folds compared to the control cells (Fig. 6A). Therefore, it was found that specific caspase-9 inhibitor Z-LEHD-FMK and specific caspase-3 inhibitor Z-DEVD-FMK could block the apoptosis induced by AuNPs.

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Fig. 6

The effects of 1.5 µM greenly synthesized AuNPs alone or in combination with specific caspase-9 inhibitor Z-LEHD-FMK or specific caspase-3 inhibitor Z-DEVD-FMK on (A) apoptosis, (viability) of human cervical cancer HeLa cells after 24 h. **P < 0.01, ***P < 0.001 compared to control. ^$P < 0.05, ^$XXX^$P < 0.01 compared to HeLa cells.

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As intrinsic apoptosis induction and relevant cell death are mediated by the upregulation of caspase-9 and caspase-3, we also evaluated the rate of viable cells by MTT assay. Fig. 6B shows that although the incubation of HeLa cells with 1.5 µM AuNPs resulted in a significant reduction in cell viability after 24 h, pre-treatment of cells with caspase-9 and caspase-3 inhibitors, significantly recovered cell viability. Taken together, these data indicated that greenly synthesized AuNPs induced caspase-mediated apoptosis through the mitochondrial pathway in HeLa cervical cancer cells.