Biofabrication of silver nanoparticles from aqueous leaf extract of Leucas aspera and their anticancer activity on human cervical cancer cells

Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), and dimethyl sulfoxide (DMSO), antibiotics (penicillin and streptomycin), propidium iodide (PI), ethidium bromide (EtBr) were purchased from HiMedia Laboratories (Mumbai, India). Quercetin standard was purchased from Sigma-Aldrich (St. Louis, USA). Sodium pyruvate, NADH was purchased from SRL Chemicals (Mumbai, India).

The HeLa cell line was procured from Tamil Nadu veterinary and animal sciences university (Chennai, India). Cells were cultured in DMEM medium with 10% of fetal bovine serum and 1 ml of antibiotic solution, and then incubated at 37 °C in humidified CO₂ incubator.

The Leucas aspera leaves were collected from SRM Institute of Science and Technology campus, Chennai and shade dried for a week for removing moisture from the sample. Then, it was subjected for grinding using mixer grinder. After sieving, uniform size of fine powder was obtained and stored in container for further studies.

Synthesis of nanoparticles was performed using aqueous plant extract as described previously [22]. Briefly, 5 g of leaf powder was mixed with 100 ml of Millipore water. The solution was boiled for 10 min at 60 °C in the water bath and filtered using Whatmann filter. The filtrate collected was stored at 4 °C. Briefly, 45 ml of 1 mM silver nitrate solution was prepared, and 5 ml of aqueous leaf extract was added dropwise on constant stirring. The solution was then left on constant stirring to observe the colour change, which indicates the reduction of silver. The solution was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and the pellet was stored for further use at 4 °C.

The UV-Visible spectral analysis was performed for green synthesised nanoparticle according to [23]. Briefly 4 ml of the reaction mixture was taken in the quartz cuvette for spectral analysis. The spectroscopic readings were taken at different time intervals of incubation (30, 60, 120, 150 and 180 min). The spectral analysis was estimated using UV3600-Shimadzu UV-vis-NIR spectrophotometer with wavelength ranging from (300 nm–700 nm).

The green synthesise silver nanoparticles were investigated using zeta potential analyser to measure particle size and surface charge. The nanoparticle pellet was re-suspended in Millipore water and alalysed using Horiba Scientific nanoparticle analyser (SZ-100), Japan [24].

The green synthesised nanoparticles were subjected to scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) to determine the morphological size, shape and elemental composition of synthesised nanoparticle [25]. The analysis was performed using the Ultra 55 Model-II Carl Zeiss SEM, Germany.

The green synthesised nanoparticle was investigated using FTIR analysis to determine the reactive functional groups involved in the interaction between the plant leaf extract and silver nitrate. The green synthesised nanoparticle pellet was re-suspended with 1 ml of deionised water and grounded with KBr pellet. Then, it was subjected to FTIR analysis using Agilent Technologies carry 660 FTIR spectrometer. The functional groups were identified using the reference peak values [26].

Cells with a density of 0.5 × 10⁵ were seeded in 24 well plates containing 1 ml of DMEM and incubated at 37 °C in humidified CO₂ incubator for 24 h for cells to attach. After incubation, the cells were treated with three different concentrations of green synthesised nanoparticles (50 μg, 100 μg, 150 μg) and incubated for overnight. The untreated wells served as negative control whereas the quercetin treated cells served as positive control. After 24 h incubation, 50 μl media was transferred to 2 ml of LDH buffer and incubated in dark at room temperature for 30 min and substrate was added freshly before taking reading and absorbance was measured at 340 nm.

Morphological changes of cervical cancer cells after green synthesised nanoparticles treatment were determined using phase contrast microscopy. The cell culture plate was treated with different concentrations (50, 100 and 150 μg ml⁻¹) of green synthesised silver nanoparticles and incubated for 24 h in CO₂ incubator. Then, the cell morphological changes were determined using phase contrast microscopy [27]. The images were captured using Olympus inverted microscope.

The green synthesised silver nanoparticle treated cells (3 × 10⁶) were trypsinised and suspended in 180 μl of lysis buffer (150 mM NaCl; 10 mM EDTA; 50 mM Tris.Hcl; 1% SDS) and incubated on ice for 20 min. After incubation 20 μl of RNase (20 mg ml⁻¹) was added and incubated for 1 h at 37 °C. Further, 20 μl of proteinase K (20 mg ml⁻¹) was added and incubated at 56 °C for 2 h. Then, DNA was separated using phenol-chloroform and isolated DNA was stored at −20 °C. The DNA samples were resolved in 1.5% agarose gel and visualised under gel documentation system (Biorad, USA) [28].

After green synthesised silver nanoparticles treatment, cells (1 × 10⁶) were trypsinised and suspended in PBS and centrifuged at 1200 rpm for 5 min at room temperature. The supernatant was aspirated and pellet was re-suspended with 500 μl of PBS and washed again. The cells were fixed by adding 4.5 ml of ice cold ethanol and the tubes were incubated at −20 °C for one hour. Then, the suspension was centrifuged at 1500 rpm for 5 min and the supernatant was discarded. Cells were washed again with 5 ml PBS and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and cells were suspended in 1 ml of propidium iodide (50 μg ml⁻¹), and incubated in the dark for 30 min at room temperature [29]. Finally, the cells were analysed with BD FACS calibre flow cytometer (BD Biosciences, San Jose, CA) to determine apoptosis.

The statistical analysis of data obtained was performed using the software Graph Pad Prism 7.0 for Windows. All data obtained were represented as a mean ± standard error of the mean. One-way analysis of variance (ANOVA) was used for statistical analysis.

In the present study we demonstrated the effect of green synthesised silver nanoparticles on HeLa cells. The silver nanoparticles were reduced with the aqueous leaf extract of Leucas aspera. Previous studies have shown that the Leucas aspera exhibits immunomodulatory, anti-inflammatory and anti-oxidant properties and also reported that it inhibits the prognosis of cancer. It has also been reported in another study that it shows anticancer activity in lymphoma [14]. The organic chemicals present in the aqueous extract of Leucas aspera acts as a reducing and stabilising agent for synthesis of silver nanoparticles. The mixture of silver nitrate turned brownish from yellow after 45 min at 50 °C, which represents the reduction of silver to a nanoparticle as shown in figure 1.

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3.2.1. UV-Visible spectroscopy

After nanoparticles synthesis, the solution was analysed using UV-Visible spectroscopy to determine the shift in plasmonic resonance of silver. The shift was observed at absorbance peak of 450 nm as depicted in figure 2.The reduction of silver nitrate was clearly visualised in the spectral graph of UV-Visible spectroscopy. A shift in the absorbance peak was formed around 450 nm which infers the optimal reduction and formation of silver nanoparticles as reported in the previous study [23]. It was also reported that in the synthesis of silver particles by plant extracts, shift in the absorbance peak was formed at 450 nm [30] which, clearly demonstrates the nanoparticle synthesis. This shift in the absorbance peak is due to the plasmonic resonance of silver. The peak showing in this range confirms the synthesis and presence of AgNPs.

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3.2.2. Dynamic light scattering and zeta potential analysis

The green synthesised nanoparticles were further characterizsed by different techniques. Dynamic light scattering was performed to determine the size of the nanoparticles and zeta potential analyser was used to determine the surface charge of the nanoparticles. The mean diameter was found to be 200 nm as depicted in figure 3(a) and the zeta potential was measured as surface charge, which was around −55.1 mV as depicted in figure 3(b). The surface charge of the particle was −55.1 mV which is inferred to be good. If surface charge is more negative, it can promote optimal interactions with biomolecules. Similar type of result was obtained during the synthesis of AgNPs using Urtica dioca aqueous extract which infers the formation of silver nanoparticles [31]. Another study also reported that aqueous extract of Pedalium murex used for the synthesis of AgNPs has also shown similar type of results [32].

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3.2.3. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS)

SEM is a powerful tool to analyse the surface and shape of the nanoparticles. Scanning electron microscopy is used to determine the particle size of the green synthesised silver nanoparticles, which was found to be 50 nm (figure 4). The particles under the range of 100 nm were considered nano and called nanoparticles [33]. The dynamic light scattering was used to determine shape of the green synthesised silver nanoparticles, which was found to be 200 nm which can be because of particle aggregates and formed complexes of nanoparticles. However, the result of scanning electron microscopy confirms the size of the nanoparticles. In addition the nanoparticle was also investigated with energy dispersive spectroscopy to determine the atomic elements in the nanoparticle as shown in figure 5. The silver was discovered in the spectra at 3 keV with an atomic percentage of 55.32 [5]. Previous studies have also reported that the nanoparticle size was around 25 nm–80 nm which is an optimal size for drug delivery [22] and energy peaks in the range of 2 KeV and 4 KeV [25].

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3.2.4. FTIR analysis

FTIR spectroscopy is used to identify the presence of different functional groups that are present in the sample mixture by calculating the vibrational frequencies of the chemical bonds between the atoms [26]. Green synthesised silver nanoparticles were analysed by FTIR to determine the functional groups present in the synthesised nanoparticles. The elemental composition was interpreted using the software IRpal 2.0 and the results were similar to the previously reported studies as depicted in figure 6(a). Functional groups belonging to classes of carboxylic acids, alkynes etc, were identified. In present study FTIR analysis was applied to determine the functional group involved in the reduction of Ag⁺ to Ag⁰ and capping of biologically reduced silver nanoparticles synthesised using Leucas extract. The broad and wide peak existing at 3343 cm⁻¹ as shown in figure 6(b) is specified for O–H functional group ensuring the presence of phenolic group and flavonoids [5]. The sharp broad absorption peak at 1636 cm⁻¹ related to C=N indicating the presence of azomethines. They are considered under imines and aldimines. These results support that the compounds present in green synthesised AgNPs provide the stable interaction between plant and silver nanoparticles. The average intensity band seen in the region at 668 cm⁻¹ represents C–H stretching of alkane region. Furthermore the band at wavenumber 549 cm⁻¹ indicates the presence of C–Br functional group belongs to alkyl halides. Eventually, the result supports that presence of flavonoids/phenolic compounds acts as a reducing and stabilising agent.

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The green synthesised silver nanoparticles were treated to cancer cells and their cytotoxic effect was investigated. To determine the cytotoxicity of green synthesised nanoparticles on HeLa cells, cells were seeded in 24 well plates at a density of 50,000 cells per well and incubated for 24 h for the attachment. Cells were treated with different concentrations (50 μg, 100 μg, 150 μg) of nanoparticles and after 24 h incubation, LDH assay was performed. Lactate dehydrogenase is an enzyme which is present in animal cells that gets released when cells undergo damage or injury [34]. The LDH release from the damaged cells was measured to determine the cytotoxicity of the nanoparticles. The quercertin was used as a positive control. The obtained results showed that the increasing drug concentration increases the release of LDH from treated cells which infers to dose dependent toxicity shown in figure 7. The cytotoxicity (58%) was observed for the concentration of 150 μg ml⁻¹. The increased release of LDH is the direct proportionality for the toxicity of cells. So, the concentration which has the highest LDH release is highly toxic [35]. A significant validation of LDH release was reported for AgNPs synthesised from Chrysanthemum indicum against 3T3 mouse embryo fibroblast cells [36].

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A dose dependent morphological change in the cell culture plate treated with the green synthesised silver nanoparticles was observed through phase contrast microscopy. Dysplastic morphology with cell blebs was seen along with the increased cell death in correspondence to increase of concentration of nanoparticle treatment which is shown in figure 8. At lower concentrations, cells remained intact with less dead cell population, whereas at higher concentrations the number of dead cells increased along with the cell shredding, which indicates that the cell death is the result of apoptosis. The cells were incubated with drug for 24 h before they were visualised under microscope. Similar results were reported in LoVo cells when treated with AgNO₃ nanoparticles synthesised by the reduction of plant extract from Ferula asafoetida [27].

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The cells were treated with green synthesised silver nanoparticles to determine the induction of DNA damage. The cells were treated with 150 μg ml⁻¹ drug concentration and incubated for 24 h. The visualisation of isolated DNA in agarose gel shows the formation of DNA ladder as depicted in figure 9 which indicates the occurrence of apoptosis [37]. Apoptosis is the major signal to maintain homeostasis in the body [38]. Induction of apoptosis results in the cleavage of DNA. The physiological characteristics of apoptosis include the membrane flipping, DNA damage, apoptotic bodies [39]. In the present study the cells were treated with synthesised silver nanoparticles resulting in the induction of apoptosis. DNA isolated from untreated cells was used as a negative control, and the 1Kbp ladder was used as a marker. The results that were obtained showed that green synthesised nanoparticles induced the DNA damage at 150 μg ml⁻¹ concentration. The reports have shown that the synthesised biogenic nanoparticles induce apoptosis more efficiently than control [38]. The DNA was fragmented at the given concentration that infers the induction of apoptosis. Similar kind of band patterns is visualised in the investigation of toxic effect of AgNPs synthesised from Solanum nigrum against hepatic cells [40].

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The agents that have ability to induce apoptosis also possess the ability to inhibit cell growth [36]. To determine effect of nanoparticles on cell growth, cell cycle analysis was performed using flow cytometry. Control cells and cells were treated with different concentrations (50 and 150 μg ml⁻¹) of nanoparticles. After incubation, they were stained with propidium iodide. In this study, different phases of the cell cycle were analysed in treated cells and control cells. G1 and G2 phases are responsible for protein synthesis and S phase for DNA replication [27]. Obtained results showed that cells treated with nanoparticles induce apoptosis. Green synthesised silver nanoparticle completely arrested the cell cycle and results have suggested that the nanoparticles inhibited the cell growth at almost every phase of cell cycle, i.e., G0/G1, S, G2/M phases as shown in figure 10. Similar kind of results was observed in the investigation of cytotoxic effect of biosynthesised AgNPs using Nepeta deflersiana against the human cervical cancer cell lines (HeLa) [41].

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