Introduction

Nanotechnology is commonly defined as the science of nanoscale dealing with the particles in the range of 1 to 100 nm with considerable potentials in different fields including biology, medicine, food, physics, chemistry, electronics, etc. [1, 2]. Nanotechnology provides new opportunities for pharmaceutical applications due to the specific behavior of nanomaterials compared to their parent bulk [1, 3, 4]. Recently, AuNPs have attracted highlighted interest for their significant anticancer activities [5, 6]. AuNPs have unique physiochemical properties namely large scale specific surface, high surface activity, powerful antioxidant capacity, superior biocompatibility as well as SPR [7]. Binding affinity of AuNPs towards amine and thiol groups allows their surface modification [8]. These properties enable them to be used in several biomedical applications and their exploitation at the molecular level [7, 8]. Due to the presence of a negative charge on AuNPs, they can be easily functionalized by a vast range of biomolecules, such as drugs, genes and targeting ligands [9]. Additionally, AuNPs offer for the advantages of being nontoxic [10].

Nineteenth century marked the advent of AuNPs formation when Michael Faraday published the first scientific paper on AuNPs synthesis describing the production of colloidal gold by the reduction of Tetrachloroauric acid [11]. Several experiments were being attempted by different researchers for understanding the consequence of temperature and pH on synthesis of AuNPs, formed via hydroxyl-o-sulfonic acid reduction and to graft other biomolecules such as transferrin as well as testing these AuNPs-biomolecules conjugates in biological applications [12]. But, the green synthesis approach are nontoxic, one step, easily available in affordable value and thus are more preferable. The green synthesis approach contains the use of natural resources such as plant extracts and microorganisms as reducing and stabilizing agents for fabrication of nanoparticles [13,14,15,16,17,18,19,20,21,22].

The Pongamia pinnata plant used in the present study belong to the family Fabaceae and subfamily Papilionaceae [23]. Phytochemical constituents of this plant includes flavonoids, flavones, furane, furanoflavonols, chromenoflavones, furanodiketones, flavonoid and glycosides [24, 25]. Due to presence of these secondary compounds, P. pinnata extract have potential to be used in biological applications such as antioxidant, antiproliferative, anti-inflammatory and anticancer agent [26]. Different parts of Pongamia plant serve as excellent crude drug for tumors [27, 28]. Recently, the potential of P. pinnata as an effective antimicrobial and therapeutic agent has been reported [29,30,31]. It has been reported that plant biomolecules could serve as a ready source of reducing and stabilizing agent for the synthesis of a variety of nanomaterials [32, 33]. In the current approach, we communicate green synthesis of AuNPs by a fast, one step, and ecofriendly method using the leaf extract of P. pinnata, a plant of medicinal importance, which served as both reducing and stabilizing agent.

Although there are various effective techniques available for the treatment of cancer such as surgery, chemotherapy, radiation therapy, vaccinations, immunotherapy, photodynamic therapy, stem cell therapy [34], the worldwide incidence rate and mortality rate of cervical cancer still remains much higher (ranks fourth in both, among females) compared to other types of cancers [35]. Cervical cancer has second most incidence and mortality rates among women in most of the low/medium HDI countries [35]. In India only, cumulative lifetime risk and cumulative death risk from cervical cancer in women are 2 and 1%, respectively [36]. Cellular processes such as generation of ROS and loss of MMP are linked with the execution of the apoptosis pathways of cell death [37]. Treating cancer by induction of apoptosis using various plant chemotherapeutics is one of the most fascinating new approaches in cancer research [38]. In the present study, not only an in-depth characterisation of the synthesised AuNPs was performed, but also, considering the significant pharmacodynamics properties of AuNPs, a comprehensive investigation was performed to explore their resonate antiproliferative properties and the possible molecular mechanism involved in induction of cytotoxicity in HeLa cells.

Materials and Methods

Chemicals

Tetrachloroauric acid (HAuCl4·3H2O), DMEM, fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), H2DCFDA, Rhodamine 123, and Propidium Iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). All others chemicals were of analytical grade. All glassware used in the study were properly rinsed with double distilled (DD) water before their use. The stock solutions of the chemicals were made in DD water or other appropriate solvent. Besides, fresh leaves of P. pinnata were collected from Jawaharlal Nehru University Campus, New Delhi, India.

Cell Culture

HeLa and HEK 293 cell lines were obtained from National Centre for Cell Science, Department of Biotechnology, Pune, India. The cells were cultured in DMEM medium supplemented with 10% (v/v) FBS, antibiotics (streptomycin 10 µg/mL, penicillin 100 U/mL). Cells were trypsinized, sub cultured and allowed to grow till confluency. Cells were maintained at 37 °C in 5% CO2 atmosphere in a humidified incubator.

Synthesis of AuNPs

Preparation of Plant Extract

Paullinia pinnata leaves were collected from JNU campus, thoroughly washed with DD water and left to dry at room temperature (RT). For preparing plant leaves extract, 10 g of leaves were weighed, boiled in 100 mL of DD water for 30 min under constant stirring. The obtained plant extract was filtered through Whatman filter 41 and stored at 4 °C for further experiment.

Preparation of AuNPs

PLE was appropriately diluted plant extract prior to synthesis. The diluted extract was boiled and stock solution of HAuCl4 was added to the PLE solution in varying concentration. The reaction mixture was boiled till a change in colour of the solution was observed. The colloidal suspension was filtered through 0.22 µm filter and stored at 4 °C for further analysis.

Characterisation of AuNPs

The phase purity and crystalline nature of the AuNPs were determined by various analytical characterisation techniques. UV–Visible absorption spectra was recorded on an Agilent (UV 2300) double beam spectrophotometer in the range of 200 to 800 nm. Particle size was analyzed by Transmission electron microscopy TEM (JEOL) 2100F TEM instrument, operating at a voltage of 200 kV. The HR-TEM analysis of AuNPs was done by placing a drop of nanoparticle solution on to carbon-coated copper grids. For XRD analysis, the colloidal suspension was drop coated on to glass slides and dried to prepare thin films. Readings were taken on a Seifert X-ray diffractometer. The instrument worked at 40 kV with tube current 30 mA under Cu-Kα radiation, in the peak range of 10–90. Hydrodynamic radius (Rh) of particles was estimated by dynamic light scattering (DLS) instrument (Photocor Instrument, USA) and calculated using the Stokes–Einstein equation. Scanning Electron Microscopy (SEM)-Energy-Dispersive X-ray Spectroscopy (EDX) studies were performed on LYRA3 (TESCAN).

Cell Viability Assay

Evaluation of the anticancer potential of AuNPs was done by MTT cytotoxicity assay. HeLa and HEK293 cell lines were used for the study. Briefly, 5 × 103 cells were cultured in 96 well plate and incubated overnight. On completion of incubation period medium was aspirated and cells were treated with varying concentration of AuNPs (20, 50, 100 and 200 µg/mL) for 24 h. Media were removed and 30 µL of 1 mg/mL MTT (Thiazolyl Blue Tetrazolium Bromide) and 70 µL fresh medium was added to each well and incubated for 4 h. Then, reaction mixture was aspirated and the resulting formazan crystals were solubilized in 100 μL DMSO. Absorbance was taken at 595 nm in multi-mode microplate reader (SpectraMax M5-Molecular Devices, USA). Untreated sets served as control that was run simultaneously under similar conditions. Cell inhibition percentage was calculated using the following formula.

$$ \% Cell\;inhibition = \frac{{Mean\;OD \left( {Control\;cells - Treated\;cells} \right)}}{Mean\;OD\;of\;control\;cells }*100 $$

The IC50 values for all AuNPs were obtained by scheming percentage inhibition versus concentration of the AuNPs.

Morphological Analysis

For the study, approximately 1 × 105 cells were cultured in a 6 well plate and incubated overnight followed by treatment with 200 μg/mL of synthesised AuNPs-S1, S2, and S3. EA-Prime Inverted phase contrast microscope (Lmi microscopes, UK) was used to analyse the morphological alteration after 24 h and images were captured at 10× magnification.

Flow Cytometric Analysis for ROS Generation

Effects of AuNPs on intracellular ROS generation in HeLa cells was analyzed by flow cytometry using a cell permeable and free radical indicator H2DCFDA (2′,7′-dichlorofluorescein diacetate). Intracellular de-esterification of non-fluorescent H2DCFDA convert it to highly fluorescent 2′,7′-dichlorofluorescein (DCF) upon oxidation. This assay was performed following a protocol with some modification as previously described [39]. Briefly, 2 × 105 cells were cultured in 6 well plate incubated to grow overnight. Spent media was discarded and 200 μg/mL of AuNPs was added and incubated for 24 h. Then, cells were harvested, washed with PBS, reincubated with 10 μM H2DCFDA in serum free medium for 45 min in dark. Cells were washed twice with cold PBS and fluorescence intensities of 10,000 events of each sample was recorded in triplicates immediately by flow cytometer, BD FACS Verse in FITC Channel. The data were analysed using BD FACSuite software.

Mitochondrial Membrane Potential Assay (Δψm)

Rhodamine 123 was used to assess changes in MMP due to treatment of AuNPs. Rhodamine 123, a cationic lipophilic fluorochrome gets selectively permeabilised and retained inside mitochondria with intact membrane potential. Loss of MMP causes leakage of Rhodamine out of the cell. Hence, measurement of fluorescent intensities of Rhodamine 123 is correlated with the Δψm. MMP in HeLa cells after AuNPs treatments was determined using Flow cytometry following a protocol described earlier with some modifications [40]. Briefly, 2 × 105 cells were seeded in 6 well plate, incubated to attain required confluency. The subsequent day, cells were exposed to 200 μg/mL of AuNPs and allowed to interact for 24 h. Cells were harvested, washed with PBS and then loaded with 10 μg/mL of Rhodamine 123 (Rh-123) in serum free medium for 30 min in dark. Then, cells were washed twice with cold PBS and fluorescence intensities of 10,000 cells of each sample in triplicates were acquired immediately using flow cytometer, BD FACS Verse in FITC Channel and data were analysed using BD FACSuite software.

Wound Healing/Cell Migration Assay

Wound healing assay was conducted as previously described with some modifications [41]. Briefly, Approximately 4 × 105 cells were seeded per well in six well plates. When cells attained 80–90% confluency, a wound was made in each well via a sterilized 200 μL tip and spent media were replaced with new media containing 200 μg/mL of AuNPs. Images of wound were taken at 0 h, 24 h and 36 h using an EA-Prime inverted phase contrast microscope (Lmi microscopes, UK) and percentage decrease in wound width (thickness) were calculated.

Cell Cycle Analysis

Cell cycle analysis was performed by following a protocol as previously described with some modifications [42]. Briefly, 2 × 105 cells were added into each well and incubated for 24 h. Cells were treated with and without 200 μg/mL of AuNPs for 24 h. Then, cells were collected by trypsinization and washed twice with cold PBS by centrifugation at 1000 rpm. Cell pellet was resuspended in PBS, fixed with chilled ethanol (70% v/v) and kept at 4 °C overnight. Cells were thereafter washed with cold PBS and stained with staining buffer containing Propidium Iodide (50 μg/mL), RNase (40 μg/mL), 0.1% sodium citrate and 0.03% Triton X 100 for 30 min at 37 °C in water bath in dark. Then, 10,000 events of each samples were analyzed on BD FACS Verse flow cytometer. During analysis, aggregates were removed by applying gate for doublet elimination. The percentage of cells in G0/G1, S, G2/M and SubG1 phases of the cell cycle was determined using ModFit LT software (Verity Software House Inc., USA).

Statistical Analysis

All experiments were performed three times, each in triplicates. Mean ± SD was determined. Significance of data was tested by one way ANOVA followed by Tukey’s post hoc multiple comparison test with p < 0.05 Vs Control using GraphPad Prism 5 software for Windows (San Diego CA, USA).

Results and Discussion

Synthesis and Characterisation of AuNPs

We used fresh PLE for synthesis of colloidal AuNPs via a feasible, one-step and green method. Different size of AuNPs were formed by varying the initial precursor ion concentration. Scheme 1 shows the graphical representation of AuNP formation from P. pinnata leaves extract. We used the leaf extract of fresh P. pinnata for biosynthesis of AuNPs. The approach used was green synthesis protocol using an aqueous solvent for synthesis. Prior to synthesis, leaf extract was diluted in 5 mL of DD water and boiled. Upon boiling, HAuCl4 at varying concentrations were added to the solution and reaction was stopped on observation of change in colour of solution. Appearance of a reddish/purple colour indicated the formation of AuNPs. Control set in which the PLE only were boiled, did not show any change in colour. This suggest that the components of PLE were capable of serving the dual role of reducing and stabilizing agent, as the addition of HAuCl4 resulted in formation of colloidal AuNPs. Table 1 summarizes the amount of precursors used for AuNPs synthesis of different sizes.

Scheme 1
scheme 1

Graphical illustration showing the preparation of PLE and AuNPs using the metal (Au3+) precursor, HAuCl4 that is reduced to (Au0) by the secondary metabolites present in PLE

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Table 1 Summary showing the variation in HAuCl4 concentration for optimization of AuNPs formation
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UV–Visible Spectroscopy

Change in color of extract solution from light-green to reddish/purple and the presence of the characteristic plasmon peak in the UV–Visible spectrum in the range of 525–540 nm marked the formation of AuNPs. This peak is typical to spherical AuNPs with a 10–18 nm long diameter [43,44,45]. AuNPs formation was confirmed by visualizing kinetics of reaction via UV–visible spectroscopy and was found to take 3 min to complete their formation reaction, suggestive from Plasmon peak which showed no significant variation beyond this time. Figure 1a represent typical SPR spectra of the synthesised colloidal AuNPs.

Fig. 1
figure 1

a UV–Visible spectra of different colloidal AuNPs. b X-ray diffraction recorded from drop–coated films of AuNPs. c Scanning electron microscopy of AuNPs. d Energy-dispersive X-ray spectroscopy showing strong elemental signal for Au

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XRD Analysis

X-ray diffraction (XRD) analysis was performed for determination of crystalline nature of particles. The synthesised AuNPs shown in Fig. 1b exhibited distinct Bragg reflection peak which are indexed to face centre cubic (fcc) lattice of AuNPs at 2θ values of 38.49°, 42.21°, 64.53° and 77.39°. These lattice correspond to the characteristic reflection from lattice of (111), (200), (220), and (311) as depicted in literature study [46, 47].

SEM–EDX Analysis

SEM image of AuNPs has been displayed in Fig. 1c which shows the surface morphological feature of synthesised particles. SEM image shows the spherical nature of particles, most of which were aggregated with average size of 55 nm. Further, EDX analysis showed strong signals for Au conforming the formation of AuNPs (Fig. 1d) [48, 49].

TEM Analysis

We performed TEM analysis to determine the particles size of colloidal AuNPs and found that the particles were homogeneous in shape and size. The concentration of HAuCl4 was found to be a detrimental factor and affects the average particle size. Average size of S1, S2 and S3 was found to be 10, 12, and 18 nm, respectively. The inset in the Fig. 2 shows the HR-TEM images showing the lattice firings with a size of 0.21 nm [50, 51]. The corresponding SAED image is shown in Fig. 2a while the corresponding TEM images are shown in Fig. 2b–d. Figure 2e shows the particle size histogram for colloidal AuNPs synthesised at varying HAuCl4 concentrations.

Fig. 2
figure 2

a SAED image of colloidal AuNPs. b–d TEM images of AuNPs synthesised at 1 (S1), 0.5 (S2) and 0.25 (S3) mM of HAuCl4 concentration respectively. Inset shows the HR-TEM image of respective particles. e Graph showing the average particle size of the synthesised AuNPs. f DLS analysis showing the Rh value of particles

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DLS Analysis

We supplementary performed DLS analysis of the particles to determine the mean hydrodynamic radius (Rh) of particles. The Rh value of particles was 29.82 ± 2, 33.91 ± 1.5 and 62.12 ± 1 nm for AuNPs synthesised at 1, 0.5 and 0.25 mM HAuCl4 concentration, respectively [51]. Figure 2f shows the DLS data of obtained AuNPs.

Cytotoxicity Analysis of AuNPs

We analyzed the cytotoxic potential of formed AuNPs by MTT assay. A selective cytotoxicity of synthesised nanoparticles towards HeLa cells was observed compared to HEK293 cells (Fig. 3a, b). Besides, a dose dependent cytotoxicity was observed on increasing the AuNPs concentration. IC50 values of AuNPs were determined to be 200 µg/mL for HeLa cells. IC50 is referred to as the concentration of drug/nanoparticles treated at which 50% cell death occurs. Additionally, the AuNPs synthesised at varying HAuCl4 concentration showed a similar level of cytotoxicity which may be due to the almost similar size range of particles. Cells use receptor mediated endocytosis for internalization of minute sized particles and thus this can be the mechanism via which AuNPs were taken up by the HeLa cells in the present case [52]. As the particles possessed an average size of 10–20 nm, the internalization was similar for all three AuNPs leading to similar degree of cytotoxicity.

Fig. 3
figure 3

MTT analysis of synthesised AuNPs on a HEK 293 cells b HeLa cells. Note the difference in Y-axis scale suggesting higher cytotoxicity of particles towards HeLa cells

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Morphological Evaluation of AuNPs Treated HeLa Cells

Microscopic observation of synthesised AuNPs treated HeLa cells were performed by inverted phase contrast microscope (at 10× magnification). Altered cell morphology with many rounded and condensed cells showing cytoplasmic shrinkage was found (Fig. 4b–d). Some spiked cells were also observed (Fig. 4c). Many detached, floating and dead cells with fragmented nucleus were also observed (Fig. 4b). AuNPs named S1 was found to have maximum potential for causing cell death and growth deformities.

Fig. 4
figure 4

Cell morphology analysis in HeLa cells. Cells were treated with AuNPs for 24 h and photographs were captured using inverted phase contrast microscope at 10× magnification a control with normal morphology. b Cells treated with S1 showed many floating and dead cells; purple arrow indicates cells with cytoplasmic shrinkage, yellow arrow indicates nuclear condensation, light blue arrow indicates nuclear fragmentation and red arrow indicates formation of apoptotic body. c Cells treated with S2; green arrow indicates spiked cells. d Cells exposed to S3

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Effect of AuNPs on Cell Migration

In vitro wound healing or cell migration assay is a simple but an important assay in order to investigate the migration potential of a treated cell as compared to untreated cell. Thus, it is helpful in determining the effectiveness of a strategy to inhibit cell growth. This assay was also performed on HeLa cells to determine the wound healing capacity of the synthesised AuNPs due to migration of cells from the edges of the created wound in comparison to untreated cells. As shown in Fig. 5a, reduction in wound width after 24 h of HeLa cells exposure to AuNPs named S1 was non-significant whereas reduction brought about by S2 and S3 were found to be statistically significant (p < 0.01). After 24 h of treatment, S1, S2 and S3 decreased the wound width by 8.12, 37.93 and 19.39%, respectively as compared to 59.10% decrease in control. It indicated that S1 possess the maximum inhibitory potential towards HeLa cell migration among AuNPs.

Fig. 5
figure 5

Effect of AuNPs on in vitro wound healing property of HeLa cells determined by measuring wound thickness (width). Wounds were made using sterile 200 μL tip and images were captured at 0 h, 24 h and 36 h by inverted phase contrast microscope at 10× magnification. a Representative images of three independent experiments, showing variation in wound width after treatment with AuNPs–S1, S2 and S3 for 24 and 36 h as compared to control. b corresponding bar diagram indicating significance level of these changes in wound width; arrows indicate the migrating HeLa cells. Data are represented as the mean ± SD separate experiments conducted in triplicates. Significance of data was tested by one way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Asterisks over bar represents level of significance between the indicated groups

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Effect of AuNPs on Generation of Intracellular ROS

In order to investigate whether green synthesised AuNPs induce intracellular ROS generation and subsequent change in ROS associated signalling leading to cell death in HeLa cells, H2DCFDA—a sensitive and highly fluorescent probe was used to measure the intracellular ROS levels. Histogram representing mean fluorescent intensities (MFIs) showed a rightward shift in the MFI peaks of each sample (Fig. 6a). Thus, a significant (p < 0.001) increase in ROS production with respect to control was observed for all three AuNPs upon treatment with 200 μg/mL for 24 h in HeLa cells. Further, out of the three AuNPs, S1 caused maximum increase in ROS levels as compared to control, followed by S3 and S2 (Fig. 6b). These results were in line with findings from previous experiments which indicated that the decrease in viability and inhibition of growth of HeLa cells after 24 h, AuNPs exposure can be due to the ROS mediated induction of cell death [53].

Fig. 6
figure 6

Intracellular ROS generation in HeLa cells detected by fluorescent probe H2DCFDA using flow cytometry. a Representative histogram showing change in MFIs of each sample after treatment with AuNPs–S1, S2 and S3 for 24 h as compared to control. b corresponding bar diagram indicating significance level of change. MFI mean fluorescence intensity, PC positive control (0.5 mM H2O2, 2 h); Data are expressed as the mean ± SD of 3 separate experiments performed in triplicates. Significance of data was tested by one way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Asterisks over bar represents level of significance between the indicated groups

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Effect of AuNPs on Mitochondrial Membrane Potential (Δψm)

Increased concentration of intracellular ROS in a cell can cause collapse of MMP due to change in the mitochondrial membrane permeability [54]. Alteration in MMP is an important event associated with determination of probable mechanisms and pathways of cell death. MMP of healthy cells remain well preserved but loss of mitochondrial transmembrane potential and subsequent release of apoptotic proteins such as cytochrome c and AIF can lead to programmed cell death/apoptosis [55]. Hence, we determined the status of MMP after treatment of green synthesised AuNPs in HeLa cells by measuring the fluorescence of Rhodamine 123 in a flow cytometer (Fig. 7a). All three AuNPs significantly altered the MMP and caused the loss of MMP in HeLa cells in the order of S1 (p < 0.001) > S3 (p < 0.001) > S2 (p < 0.01) compared to untreated cells (Fig. 7b).

Fig. 7
figure 7

Effect of AuNPs on mitochondrial membrane potential detected by fluorescent probe Rhodamine 123 using flow cytometry. a Representative histogram signifying change in MFIs of each sample after treatment with AuNPs–S1, S2 and S3 for 24 h as compared to control. b corresponding bar diagram indicating significance level of change. MFI mean fluorescence intensity; PC positive control (0.1 μM Rotenone, 24 h); Data are expressed as the mean ± SD experiments conducted in triplicates. Significance of data was tested by one way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 vs control, ns non-significant. Asterisks over bar represents level of significance between the indicated groups

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Cell Cycle Analysis

We examined whether changes in the cell cycle profile was involved in the decreased viability and inhibited growth of HeLa cell after exposure to green synthesised AuNPs. Analysis of cell cycle profile (Fig. 8e) obtained by flow cytometry of cells treated with 200 μg/mL of these AuNPs for 24 h indicated that they brought significant changes in the distribution of cells in different phases of the cell cycle as compared to control (Fig. 8a). AuNPs S1, S2, and S3 decreased the percentage of G1 cells by 10.38, 12.83 and 8.68% and G2/M phase cells by 6.35, 5.34, and 7.32%, respectively as compared to untreated cells. They also induced the accumulation of S-phase cells by increasing their number from 20.3 to 37.7, 36.7, and 36.9%, respectively and thus caused S-phase arrest in HeLa cells (Fig. 8a–c). Generally, cells having hypodiploid DNA content (represented by Sub-G1 peak) were considered as the apoptotic cells [56]. Induction of apoptosis and Sub-G1 arrest in HeLa cells by these three green synthesised AuNPs was also detected as evident from the significantly high percentage (increase by 33.23, 22.77, and 27.27%, respectively against 4.85% of control) of Sub-G1 cells.

Fig. 8
figure 8

Effect of AuNPs on Cell cycle profile of HeLa cells. Cell cycle analysis was performed using PI staining by flow cytometry in BD FACS Verse flow cytometer. Representative cell cycle histogram of the HeLa cells treated with 200 μg/mL of AuNPs- a control, b S1, c S2, and d S3 for 24 h indicated S-phase arrest and Sub-G1 arrest with significant increase in S-phase and Sub G1 population as compared to control. e Bar diagram compares variation in cell distribution percentage in each phases of cell cycle of HeLa cells after AuNPs treatment as compared to control. Data represented as mean ± SD of 3 independent experiments conducted in triplicates. ANOVA followed by Tukey’s multiple comparison test was performed to test significance of data. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Asterisks over bar represents level of significance between the indicated groups

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Mechanism of Cytotoxicity

The probable mechanism of cytotoxicity of the green synthesised AuNPs in HeLa cells has been depicted in Scheme 2. AuNPs gets internalized into HeLa cells by endocytosis. Inside the cytosol, it exhibit its cytotoxic effect by elevating intracellular ROS, perturbing the MMP, arresting cells at S-phase of the cell cycle and finally inducing cell death by apoptosis.

Scheme 2
scheme 2

Schematic representation of proposed mechanism of induction of cell death by AuNPs synthesised from PLE in human cervical cancer cell line (HeLa)

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Conclusion

In the present work, we described a rapid, feasible, reproducible and eco-friendly approach for generation of AuNPs. In this approach gold ions were reduced to nanoparticles form by secondary metabolites present in P. pinnata leaf extracts. These green synthesised AuNPs have shown to cause excess production of intracellular ROS, generation of the condition of oxidative stress and subsequent loss of MMP in HeLa. Further analyses revealed that AuNPs treatment exhibited excellent anti proliferative property in the form of reduced viability and inhibition of migration in HeLa cells by arresting them at S-phase of the cell cycle and ultimately leading to induction of cell death. We also investigated that variation in HAuCl4 concentration (ranging from 0.25 to 1 mM) for better optimization of AuNPs formation does not bring much changes in the anticancer potential of these AuNPs in HeLa cells. Apart from our findings about the probable pathways of cell death, there could be possibilities of existence of other pathways of anticancer activities of these AuNPs in cervical or other cancer type both in vitro and in vivo, which further needs to be explored in order to establish them as a potential anticancer strategy. It is noted that the AuNPs surface attached phytochemicals may interfere with their anticancer effects. Overall, plant-based AuNPs have a bright future to combat cancers in the future. However, many challenges should be addressed before translating the laboratory studies for clinical trials such as acute and chronic toxicity, immunogenicity, etc.