Nanotherapy on human acute myeloid leukemia cells using RGO/Ag nanocomposites

Abstract

Nanoscale delivery systems are the newest modes of treatment as vehicles for antineoplastic agents in human hematology malignancies treatment research because of their targeting and multifunctional behaviour. The purpose of this study is to evaluate the anticancer effect of graphene oxide (GO), reduced graphene oxide (RGO), silver nanoparticles (Ag NPs) and reduced graphene oxide/silver nanocomposites (RGO/Ag) on human acute myeloid leukemia cells. In this context, RGO/Ag nanocomposites are synthesized via a green method using lactulose as a reducing as well as stabilizing agent. RGO/Ag nanocomposites have been characterized by Raman spectroscopy, thermo-gravimetric analysis and field emission scanning electron microscopy. RGO/Ag nanocomposites shows better anticancer activity than GO, RGO and Ag NPs. RGO/Ag nanocomposites exposed human acute myeloid leukemia cell show the possible contribution of apoptosis in the etiology of cell death.

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1. Introduction

Cancer has become one of the leading causes of death globally. Among different types of cancer, leukemia comprises a major cause of death in children and young adults. Acute myeloid leukemia (AML), one of the major types of leukemia, is responsible for significant death rates.¹ In AML, a group of neoplastic disorders occur which can be characterised by abnormal proliferation and accumulation of immature hematopoietic cells in the bone marrow and also in the blood circulation. It is responsible for approximately 20% of the acute leukemia in children and 80% in the case of adults.² There are various techniques available to treat this disease but the success rate is too low. Most of the conventional and traditional chemotherapeutic drugs and techniques show unidirectional side effects and are also very costly.³ For this reason, targeted cancer therapy is very necessary to reduce the harmful reactions and death rate and as well as cost involved. So, targeted therapy is gaining importance for reducing tumor size by destroying cancer cells. In this context, nanotechnology is now being introduced in the field of biomedical applications of pharmacology, bioengineering, biology and medicine.⁴

Since the discovery of graphene in 2010, researchers are trying to use this novel material in different areas of science and technology across the globe. Graphene is a two dimensional wonder material with an one atom thickness composed of sp² hybridized carbon and arranged in a honeycomb network. Graphene is used in biological applications including gene and drug deliveries. It is also used in intercellular tracking due to its potential for traversing the plasma membrane and promoting and stimulating the cellular uptake of small molecules^5,6 and macromolecules.^7,8 Graphene oxide (GO), reduced graphene oxide (RGO), few layer graphene (FLG) and ultra thin graphite all are part of the graphene family. GO is strongly hydrophilic and produces stable and homogeneous colloidal suspensions of negatively charged sheets in aqueous and polar organic mediums due to the presence of hydroxyl, carboxylic and epoxide groups on its surface. Good biocompatibility and lack of toxicity makes it a promising material for modern drug delivery applications.^5,6 GO has high efficiency loading and intelligent controlled release of multi-targeted drugs due to its high surface area. Hu et al.⁹ and Wang et al.¹⁰ reported that graphene quantum dots can act as carriers for targeted anticancer drugs and as well as DNA cleavage activity enhancers which are useful for cancer therapy.

Hummer's method is the most accepted method for the preparation of GO.¹¹ Graphene is generally synthesized by several physical or chemical methods. A chemical method for preparation of graphene needs a reducing agent as well as a surfactant like hydrazine mono hydrate, hydroquinone and hydrogen sulfide which are highly toxic to living organisms and the environment.^12,13 As a result, several green reductants as well as stabilizing agents have been developed including ascorbic acid,¹⁴ amino acids,¹⁵ glucose,¹² humanin¹⁶ and microorganisms for synthesis of graphene.^17,18

The synthesis of graphene–metal nanocomposites opens the door to investigate tunable novel properties for different applications. Incorporation of metal nanoparticles such as Ag, Au, Pt, Pd, ZnO, TiO₂, Fe₃O₄ and ZnS^19,20 into the graphene sheets generates new types of hybrid materials for possible applications in super capacitor,²¹ electro-catalysis,²² lithium ion batteries,²³ SERS,²⁴ imaging,²⁵ and also in the field of biomedical and pharmaceuticals.²⁶ GO/TiO₂ nanocomposites showed outstanding photodynamic-anticancer activities without harmful toxicity.^9,10

Silver nanoparticles (Ag NPs) have drawn the attention of many researchers in several areas including catalysis,²⁷ nanoscale electronics,²⁸ optics, energy science, imaging²⁵ and pharmaceuticals²⁶ mainly as antimicrobial agents for diagnostic purposes,²⁹ due to their superior quantum characteristics with defined and controlled shapes.³⁰

Many researchers have reported the anticancer activity of Ag NPs.³¹ Ag NPs have remarkable toxicity on various human cell lines like human lung fibroblast cells (IMR-90), human glioblastoma cells (U251),³² endothelial cells³³ and human breast cancer cells.³⁴ Individually, graphene and Ag NPs show less biological activity than graphene/Ag nanocomposites. Gurunathan et al. showed that reduced graphene oxide/silver (RGO/Ag) nanocomposites have outstanding cytotoxic effects and excellent apoptotic activity on ovarian cancer cells.³⁵ Up until this date, there is no report on the anticancer activity of RGO/Ag nanocomposites on human acute myeloid leukemia (KG-1A) cells.

In this context, we have developed RGO/Ag nanocomposites via the green route without using any hazardous and toxic reagents. For environmental concerns, we have used a simple in situ synthesis procedure where lactulose reduced graphene oxide as well as AgNO₃ were placed a pressure cooker. RGO/Ag nanocomposites were characterized by Raman spectra, TGA and FESEM. Finally, we have tried to investigate the anticancer activity of GO, RGO, Ag NPs and RGO/Ag nanocomposites in the KG-1A cell line using an in vitro model system.

2. Materials, methods and characterization

Graphite powder and lactulose were received from Sigma Aldrich Inc., St. Louis, MO. Concentrated sulphuric acid (98% H₂SO₄, GR grade), potassium permanganate (KMnO₄ purified), hydrogen peroxide solution (30% H₂O₂), sodium nitrate (NaNO₃, extra pure), and sodium hydroxide (NaOH purified) were obtained from Merck Specialties Pvt. Ltd., India. Silver nitrate (AgNO₃, extra pure) was received from Spectrochem Pvt. Ltd, Mumbai, India. RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, sodium chloride (NaCl), sodium carbonate (Na₂CO₃), sucrose, Hanks balanced salt solution, and ethylene diamine tetra acetate (EDTA), dimethyl sulfoxide (DMSO) were purchased from HiMedia, India. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent), ethidium bromide, acridine orange, 40,6-diamidino-2-phenylindole dihydrochloride (DAPI) were procured from Sigma (St. Louis, MO). Tris–HCl, Tris buffer, KH₂PO₄, K₂HPO₄, HCl, alcohol and other chemicals were procured from Merck Ltd., Mumbai, India. All other chemicals of the highest purity grade were purchased from Merck Ltd., SRL Pvt., Ltd., Mumbai, India.

2.1 Methods of RGO/Ag nanocomposites preparation

We have synthesized graphene oxide (GO) from a natural graphite powder by Hummers method.¹¹ We have done the synthesis of RGO/Ag nanocomposites in our previous work which has been published.³⁶ However, the synthesis scheme has been given in Fig. 1.

Fig. 1 Schematic diagram of RGO/Ag nanocomposites using the green method.

2.2 Characterization of RGO/Ag nanocomposites

The RGO/Ag nanocomposites were characterized in our previous research paper by X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, UV-vis absorption spectroscopy, dynamic light scattering, the energy dispersive X-ray (EDX) spectra and transmission electron microscopy (TEM).³⁶ The Raman spectra was monitored using the 1.96 eV (633 nm) line of a He Ne laser in HORIBA-JOBIN-YVON Lab RAM HR 800 instrument recorded on solid samples. Thermogravimetric analysis (TGA) was carried out using a Shimadzu TGA-50 thermal analyser in a dynamic atmosphere of dinitrogen (flow rate = 30 cm³ min⁻¹) at a rate of 10 °C min⁻¹ over a temperature range of 30–800 °C. The morphology of the synthesized sample was characterized using a field emission scanning electron microscopy (FESEM-ZEISS Auriga instrument). FESEM samples were prepared by drying a droplet of the suspension on a silicon substrate coated with gold in the sputter coater.

2.3 Methods and characterization of anticancer treatment on KG 1A (human acute myeloid leukemia) cell line

2.3.1. Cell lines culture and maintenance. The KG 1A (human acute myeloid leukemia) cell line was used for in vitro experiments. The cell line was obtained from the National Centre for Cell Sciences, Pune, India and was cultivated and maintained in a RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units per ml penicillin and 100 μg ml⁻¹ streptomycin, 4 mM L-glutamine under 5% CO₂, and 95% humidified atmosphere at 37 °C. Cells were cultured and maintained in logarithmic growth phase until number of cells reaches at 2.0 × 10⁵ cells per ml.

2.3.2. Preparation of drug. Several doses of GO, RGO, Ag NPs and RGO/Ag nanocomposites (1, 5, 10, 25, 50 and 100 μg ml⁻¹) were prepared with sterile phosphate-buffered saline (pH 7.4). A 10 mg ml⁻¹ stock for each type of particles was prepared by dispersing 10 mg of particles in PBS and sonicated for 15 minutes. It was then serially diluted using RPMI media to prepare working concentrations. In this study, all these doses were targeted against the cancer cell line for evaluation of in vitro anticancer activity.

2.3.3. Experimental design. KG-1A cells were treated with different concentrations of each type of particles (1, 5, 10, 25, 50 and 100 μg ml⁻¹) for 24 h. For a single type of particles, KG-1A cells were divided into 6 groups. Each group contained 6 Petri dishes. In every set of treatment, cell numbers were maintained to 2 × 10⁵ cells per Petri dish. The following groups were considered for the experiment and treated for 24 h.

Group I: control i.e., cells + culture media, group II: cells + 5 μg ml⁻¹ particles in culture media, group III: cells + 10 μg ml⁻¹ particles in culture media, group IV: cells + 25 μg ml⁻¹ particles in culture media, group V: cells + 50 μg ml⁻¹ particles in culture media, group VI: cells + 100 μg ml⁻¹ particles in culture media.

2.3.4. In vitro cell viability assay. The cytotoxicity of GO, RGO, Ag NPs and RGO/Ag nanocomposites were quantitatively estimated by a non-radioactive, colorimetric assay system using tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheniltetrazolium bromide (MTT).³⁷ The cancer cells were exposed to GO, RGO, Ag NPs and the RGO/Ag nanocomposite at concentrations of 0, 1, 5, 10, 25, 50 and 100 μg ml⁻¹ for 24 h, and cytotoxicity was determined using the MTT assay. The percentage of cell viability was calculated using the following equation:

% viability = [OD sample − OD control] × 100/OD control

The concentration of drug required for a 50% inhibition of viability (IC₅₀) was determined graphically. Multiple linear regressions were used to compare data using the Statistica version 5.0 (Statsoft, India) software package.

2.3.5. Intracellular uptake study. Cellular uptake of particles by KG-1A cells were studied by fluorescence microscopy according to the method described elsewhere.³ KG-1A cells (2 × 10⁵) were seeded into several 35 mm cell culture plates and were incubated with GO–RhB, RGO–RhB, Ag–RhB and RGO/Ag–RhB at their respective IC₅₀ values for fluorescence microscopy studies. After the treatment schedule, the cells were washed and collected on a glass cover slip and observed under a fluorescence microscope. Fluorescence images were taken with a 540 nm laser for differential interference contrast microscopy and 625 nm lasers for RhB excitation and emission on an Olympus research phase contrast with a fluorescence microscope (model CX40; Olympus).

2.3.6. Intracellular ROS measurement. Intracellular reactive oxygen species generation was measured by H₂DCFDA according to the method described elsewhere.³ In brief, KG-1A cell lines were treated with GO, RGO, Ag NPs and RGO/Ag nanocomposites at their respective IC₅₀ values for 24 h. After the treatment schedule, cells were washed with culture media and incubated with 1 μg ml⁻¹ H₂DCFDA for 30 min at 37 °C. Then, the cells were washed three times with fresh culture media. DCF fluorescence was observed by fluorescence microscopy (NIKON ECLIPSE LV100POL). All measurements were done in triplicate.
Pre-treatment with N-acetyl-L-cysteine. To understand the involvement of ROS in nanoparticles induced leukemic cell death, KG-1A cells were treated in a 96-well plate at 0.2 ml per well at a concentration of 2 × 10⁵ cells per milliliter. A stock solution of N-acetyl-L-cysteine (NAC; Sigma-Aldrich) was made with sterile water and added to cells at 5 and 10 mM for 1 h. After NAC pre-treatment, cells were treated with particles (IC₅₀ dose) for 48 h. Cell viability was determined by the MTT method.³⁸ All measurements were done in triplicate.

2.3.7. Cellular morphology analysis by EtBr–AO doubles staining. To understand the probable mechanism of cell death, we analyzed the cells by EtBr–AO double staining. A number of 2 × 10⁵ KG-1A cells were seeded into a 12-well plate and incubated for 24 h at 37 °C in a humidified, 5% CO₂ atmosphere. Then, all types of synthesized nano-materials were added into the well and kept for 24 h. After the incubation, cells were washed once with PBS (1×). Ten microlitres of the cells were then put on a glass slide and mixed with 10 μl of acridine orange (50 μg ml⁻¹) and ethidium bromide (50 μg ml⁻¹). The cells were viewed under a fluorescence microscope (NIKON ECLIPSE LV100POL) with 400× magnification.³⁹

2.3.8. Assessment of nuclear morphological changes by DAPI staining. The 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was performed according to the method described in our previous publication.⁴⁰ For DAPI staining, all the test cells were seeded into 12-well plates. A number of 2 × 10⁵ cells per ml were treated with or without synthesized nano-materials for 24 h and were then isolated for DAPI staining. After treatment, the cells were fixed with 2.5% glutaraldehyde for 15 min, permeabilized with 0.1% Triton X-100 and stained with 1 mg ml⁻¹ DAPI for 5 minutes at 37 °C. The cells were then washed with PBS and examined by fluorescence microscopy (NIKON ECLIPSE LV100POL).

2.3.9. Statistical analysis. All the parameters were measured in triplicate. The data were expressed as mean ± SEM, n = 06. Comparisons between the means of the control and the treated group were made by a one-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA 01060 US) with multiple comparison t tests, p < 0.05 as a limit of significance.

3. Results and discussion

3.1 Raman spectra

Raman spectroscopic analysis is a very powerful technique to investigate the crystalline nature of carbonaceous material such as graphene or graphene based materials. Fig. 2 displays the Raman spectra of RGO and RGO/Ag nanocomposites. RGO shows two prominent peaks, D band at 1330 cm⁻¹ and G band at 1594 cm⁻¹. The D band demonstrates the breathing of K point phonon A_1g, relates with the structural disorder and G band demonstrates the first order scattering of the E_2g vibrational mode of sp² carbon atoms.⁴¹ Also RGO/Ag nanocomposites show two sharp peaks, D at 1336 cm⁻¹ and G at 1594 cm⁻¹. The intensity ratio of D band/G band (I_D/I_G) for RGO is 1.23, where the same ratio of RGO/Ag is 1.30. The I_D/I_G value of RGO/Ag nanocomposites is higher than RGO due to the increase of disorder.

Fig. 2 Raman spectra of RGO and RGO/Ag nanocomposite.

3.2 Thermogravimetric analysis

Thermal stability of GO, RGO, Ag and RGO/Ag nanocomposites has been investigated using TGA, which is shown in Fig. 3. From Fig. 3, it is apparent that GO is thermally less stable than RGO, Ag and RGO/Ag nanocomposites due to the presence of oxygen containing functional groups in GO. GO starts to lose the mass from the beginning even below 100 °C due to the removal of absorbed water.⁴² At 250 °C, GO again starts losing weight due to the removal of oxygen containing functional groups. After 500 °C, the weight loss of GO has been followed by the pyrolysis of the residual oxygen containing functional groups and carbon skeleton.⁴³ RGO is more thermally stabile than GO due to the removal of functional groups during reduction. However, some functional groups are still present in RGO. Below 100 °C, RGO loses 9% of its weight due to the removal of water. However, Ag NPs are not showing any decomposition step in TGA. But after formation of hybrid materials, RGO/Ag nanocomposites show only an 8% weight loss in TGA curve below 100 °C. So, from Fig. 3 and the above discussion, it can be stated that RGO, Ag, and RGO/Ag nanocomposites are more thermally stable compared to GO.

Fig. 3 Thermogravimetric analysis of GO, RGO, Ag and RGO/Ag nanocomposite.

3.3 Field emission scanning electron microscopy

Fig. 4 displays the morphology of RGO and RGO/Ag nanocomposites. Fig. 4a and b shows the images from different sections of RGO at low and high magnification, where crumpled surface textures are present throughout the RGO sheets. FESEM images of RGO/Ag nanocomposites in Fig. 4c and d demonstrate that huge amounts of Ag NPs are well dispersed and uniformly distributed throughout the RGO sheets with the formation of an interconnected hybrid network.

Fig. 4 FESEM images of RGO and RGO/Ag nanocomposite.

3.4 Anticancer effect of RGO/Ag nanocomposites

3.4.1. In vitro cell viability assay. The anticancer activities (in vitro) of GO, RGO, Ag NPs and RGO/Ag nanocomposites have been measured towards KG-1A cell lines. KG-1A cells were exposed to different concentrations (0, 5, 10, 25, 50 and 100 μg ml⁻¹) of nano-materials for 24 h and the cell viability was measured using MTT assay. It is apparent from the results that GO, RGO and Ag NPs decrease the cell viability by 35.58%, 18.95% and 36%, respectively at a 10 μg ml⁻¹ dose, whereas RGO/Ag nanocomposites significantly decrease the viability by 46.26% using the same dose (Fig. 5). The IC₅₀ values of GO, RGO, Ag NPs and RGO/Ag nanocomposites against KG-1A cells were investigated using the Statistica version 5.0 (Statsoft, India) software package. It is clear from this statistical calculation that the IC₅₀ values of GO, RGO and Ag NPs are 18.00, 25.49 and 14.91 μg ml⁻¹, respectively, whereas, the IC₅₀ value of RGO/Ag nanocomposites is 11.58 μg ml⁻¹. RGO/Ag nanocomposites at the concentration of 11.58 μg ml⁻¹ show a significant reduction in the viability of KG-1A cells (p > 0.05 for each). The reduction in cell viability of GO, RGO, Ag NPs and RGO/Ag nanocomposites treated cancer cells occurs in a dose-dependent fashion. Thus, only respective IC₅₀ doses of each sample were selected for further experiments.

Fig. 5 In vitro cell viability assay of GO, RGO, Ag NPs and RGO/Ag nanocomposite treated KG-1A cell lines.

From these results, it is clear that the Ag and RGO/Ag nanocomposites show anticancer activity in the lowest concentration when compared to GO and RGO. In comparison to Ag NPs, RGO/Ag nanocomposites show maximum efficacy. So it may be postulated that RGO and Ag NPs in a nanocomposites system enhanced the cell killing capacity of these nanocomposites. Previously, many researchers have reported that the nanocomposite formulation is more effective in terms of anticancer activity than its isolated form.^44,45 Hence, the results of this study suggest that RGO/Ag nanocomposites may be further exploited as a potential selective anti-cancer agent.

3.4.2. Cellular ROS level. The potential for GO, RGO, Ag NPs and RGO/Ag nanocomposites to induce oxidative stress was assessed by measuring the reactive oxygen species (ROS). KG-1A cells exposed to different samples for 24 h show increased ROS formation as evidenced by the increased DCF fluorescence intensity of the images. Fig. 6A shows that GO, RGO and Ag NPs induce the ROS production individually but when RGO/Ag nanocomposites were applied in KG-1A cells, significant (p < 0.05) elevation of the intracellular ROS production was noted. ROS are molecules containing unpaired electrons and being free radicals, these are highly active and play an important role in several physiological processes including cell signalling and apoptosis leading to oxidative cell damage.⁴⁶ In healthy cells, glutathione (in its reduced form) effectively eliminates ROS and thereby maintains normal cellular metabolic behavior. The involvement of ROS has been found to be a great development in different anti-cancer research areas.^47,48 In our study, an increase of ROS levels due to RGO/Ag nanocomposite exposures may be due to the failure of the cellular anti-oxidant defense system in KG-1A cells. Not only that, the involvement of ROS can be considered as an effective contribution to apoptosis underlying the etiology of cell death.⁴⁹

Fig. 6 (A) Effects of GO, RGO, Ag NPs and RGO/Ag nanocomposite on reactive oxygen species (ROS) induction in the KG-1A cell line. Qualitative characterization of ROS formation by H₂DCFDA staining using fluorescence microscopy. (a) Control KG-1A cell line; (b) KG-1A cells treated with GO, (c) KG-1A cells treated with RGO, (d) KG-1A cells treated with Ag NPs and (e) KG-1A cells treated with RGO/Ag nanocomposite. (B) Quenching of ROS rescues KG-1A cells from particles induced cytotoxicity. KG-1A cells were pre-treated with 5 mM NAC for 4–6 h and then subsequently exposed to particles in respective IC₅₀ dose. Cell viability was estimated by MTT assay.

To reveal the mechanistic role of nanoparticles in leukemic cell death, KG-1A cells were pre-treated with 5 mM of NAC, a potent ROS inhibitor, for 4 h followed by nanoparticles exposure at their respective IC₅₀ doses for 24 h. The cell viability was estimated by MTT assay. The results demonstrated that pre-treatment with 5 mM NAC significantly protected the leukemic cells from GO, RGO, Ag and RGO/Ag nanocomposites induced cytotoxicity. Cell viability of KG-1A cells was attend at 97.25, 94.47, 95.32 and 86.84% in GO, RGO, Ag and RGO/Ag nanocomposite, respectively, compared with the control group (Fig. 6A).

From the NAC pre-treatment assay, it was found that NAC significantly protected the KG-1A cells from nanoparticles induced toxicity (Fig. 6B) by reducing intracellular ROS generation. Restoration of leukemic cell viability >86.84% in each of these cases revealed that ROS is the main causative factor whereby nanocomposites along with others particles played a mainstream anti-leukemic protective role. Intracellular elevated levels of ROS stimulated the release of various pro-inflammatory cytokines markers, including TNF-α. TNF-α activates nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (c-Jun NH₂-terminal kinase, JNK). Summative effects of the process ultimately produced cell death by apoptosis and/or necrosis.⁵⁰ To understand the probable pathway of cell death EtBr–AO double staining was performed.

3.4.3. Cellular morphology analysis by acridine orange (AO)–ethidium bromide (EtBr) double staining. Acridine orange (AO)–ethidium bromide (EtBr) double staining measures the changes of cellular morphology during cell death by apoptosis or necrosis. Fig. 7 represents the results of AO–EtBr double staining. This staining reveals that the viable cells remain intact with DNA and nuclei, and have a round and green nucleus. Early apoptotic cells have fragmented DNA, which appears as several green-colored nuclei. In late apoptotic and necrotic cells, the DNA is fragmented and has a stained orange and red color. From our results, it is clear that GO, RGO and Ag NPs did not significantly decrease the number of viable cells, but when treated with RGO/Ag nanocomposites, it significantly decreased the number of viable cells and elevated the number of early and late apoptotic cells. In case of treatment with nanocomposites, most of the cells show typical characteristics of apoptosis including plasma membrane bleeding and formation of apoptotic bodies. The number of cells stained with orange color also increased significantly. A minimum number of cells were stained red, and this indicated that most of the cells were not undergoing necrosis and that cell death occurred primarily through apoptosis.

Fig. 7 Qualitative characterization nuclear morphology by EtBr/AO dual staining using fluorescence microscopy. After the treatment schedule, leukemic cells were incubated with EtBr/AO. At the end of EtBr/AO staining, cells were washed with PBS and they were visualized by fluorescence microscopy at an excitation/emission wave length 490/620 nm. (a) Control KG-1A cell line; (b) KG-1A cells treated with GO, (c) KG-1A cells treated with RGO (d) KG-1A cells treated with Ag NPs and (e) KG-1A cells treated with RGO/Ag nanocomposite.

3.4.4. Detection of chromosome condensation by DAPI staining. Chromatin condensation was also examined by DAPI staining. When KG-1A cells were treated with synthesized nano-materials for 24 h, chromatin condensation and fragmentation were observed in the treated group (Fig. 8). The chromatin condensation and fragmentation of GO, RGO and Ag NPs show no changes or insignificant changes whereas RGO/Ag nanocomposites exhibit the highest or optimum changes in DAPI staining. Chromatin condensation/fragmentation in KG-1A cells suggest that the RGO/Ag nanocomposite significantly causes cell death by apoptotic process.

Fig. 8 Qualitative characterization nuclear morphology by DAPI staining using fluorescence microscopy. After the treatment schedule, leukemic cells were incubated with DAPI. At the end of DAPI exposure, cells were washed with PBS and they were visualized by fluorescence microscopy at excitation 330–380 nm and emission 430–460 nm. Here, (a) control KG-1A cell line; (b) KG-1A cells treated with GO, (c) KG-1A cells treated with RGO (d) KG-1A cells treated with Ag NPs and (e) KG-1A cells treated with RGO/Ag nanocomposite.

4. Conclusions

In conclusion, we summarized that RGO/Ag nanocomposites had been successfully developed by the green in situ synthesis process. Raman spectroscopy shows that the I_D/I_G value of RGO/Ag nanocomposites is higher than RGO due to the increase in disorder and defects of the carbon skeleton. RGO/Ag nanocomposites show only an 8% weight loss in TGA curve below 100 °C. FESEM images of RGO/Ag nanocomposites demonstrate that huge amounts of Ag NPs are homogeneously distributed throughout the RGO sheets. This RGO/Ag nanocomposite may be a potential candidate in various biomedical applications. The results revealed the potent anticancer activity of these nanocomposites against the KG-1A cell line compared to GO, RGO and Ag NPs. Elevation of ROS in nanocomposites exposed KG-1A cells suggests the possible contribution of apoptosis to the etiology of cell death. The EtBr–AO double staining confirmed the involvement of apoptosis rather than necrosis. So, RGO/Ag nanocomposites have great promise as anticancer agents. The application of RGO/Ag nanocomposites based on these findings may lead to valuable discoveries in the field of cancer treatment.

Acknowledgements

The author I. Roy gratefully acknowledges the Technical Education Quality Improvement Programme, University of Calcutta for providing fellowship. Dr D. Rana of Industrial Membrane Research Institute, University of Ottawa and D. Maity, B. Bhowmick, D. Mondal, G. Sarkar, N. R. Saha, A. Bhattacharyya and N. K. Bera of the University of Calcutta are acknowledged for their help. We acknowledge the CRNN, University of Calcutta for instrumental facilities.

Notes and references

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