RETRACTED ARTICLE: Dual role of ROS-dependent anti-cancer activity and photocatalytic degradation of graphene oxide against MCF-7 breast cancer cells and organic dye

Abstract

Graphene oxide (GO), a two-dimensional nanomaterial, was synthesized using a modified Hummers’ method and evaluated for its dual biomedical and environmental applications. Structural characterization confirmed the successful formation of GO, as evidenced by XRD (d-spacing of 10.23 Å), FTIR (hydroxyl, epoxy, carboxyl, and carbonyl functional groups), TGA (distinct weight loss patterns), and microscopic analyses (FE-SEM, EDX, and HR-TEM) revealing spherical and rod-like morphologies. Biologically, GO exhibited potent cytotoxicity against MCF-7 breast cancer cells in a concentration-dependent manner. The IC₅₀ concentration of the GO induced more apoptosis, nuclear membrane damage, mitochondrial dysfunction, and cytochrome c release through intracellular ROS generation, ultimately activating caspase-3 and caspase-9 pathways. These mechanistic findings were validated by fluorescence staining assays (AO/EB, DAPI, DCFH-DA, and rhodamine 123). In parallel, photocatalytic studies demonstrated that GO achieved 46.9% degradation of Rhodamine-B dye under visible light within 120 min, attributed to reduced electron–hole recombination and enhanced ROS generation. Collectively, these results highlight the multifunctionality of GO as both a promising anticancer nanomaterial targeting breast cancer and an efficient photocatalyst for environmental remediation of toxic dyes.

Introduction

Cancer remains one of the most serious global health challenges, characterized by uncontrolled cell proliferation and abnormal growth¹. Among different types of cancers, breast cancer is the most common malignancy in women and ranks as one of the leading causes of cancer-related mortality worldwide. According to the World Health Organization (WHO), breast cancer accounts for millions of new cases and deaths annually, and its impact continues to rise due to metastasis, drug resistance, and the limited efficacy of existing therapies such as chemotherapy, radiotherapy, and targeted drug delivery systems^2,3. The adverse side effects associated with conventional treatments further highlight the urgent need for safer and more effective therapeutic alternatives⁴.

In recent years, nanomaterials have emerged as promising candidates in cancer therapy due to their unique physicochemical and biological properties. Among them, graphene and its derivatives, particularly graphene oxide (GO), have attracted increasing attention. GO contains abundant oxygenated functional groups, such as hydroxyl, carboxyl, and epoxy, which enhance its hydrophilicity and allow for easy dispersion in aqueous media⁵. These properties not only facilitate drug loading and functionalization with biomolecules but also enable GO to serve as an effective nanocarrier and therapeutic agent. Several studies have demonstrated that GO can induce apoptosis in cancer cells through mechanisms such as reactive oxygen species (ROS) overproduction, mitochondrial dysfunction, and caspase activation, thereby suppressing tumor growth with minimal side effects^6–8. For example, Poongodi et al.^9,10 reported concentration-dependent cytotoxicity of GO against MCF-7 breast cancer cells, where elevated ROS levels led to nuclear damage, gene alterations, and apoptotic protein regulation.

Beyond its biomedical applications, GO has also shown great promise as a photocatalyst in environmental remediation. Photocatalytic degradation is an advanced oxidation process in which light energy activates catalysts to generate ROS, which can mineralize toxic organic pollutants into harmless end products such as CO₂ and H₂O. GO is particularly advantageous as a photocatalyst because of its high surface area, oxygenated functional groups, and excellent electron mobility. These features promote adsorption of dye molecules and improve charge separation, thereby reducing electron–hole recombination. Consequently, GO has been successfully applied for the degradation of persistent organic pollutants such as Rhodamine-B and methylene blue under visible or UV light irradiation¹¹. Its multifunctionality also allows integration with semiconductors or metal nanoparticles, further enhancing photocatalytic efficiency. Unlike most previous studies that focus either on the biomedical application or on the environmental remediation potential of graphene oxide (GO), our work uniquely demonstrates a dual functionality of GO synthesized via the modified Hummers’ method. Specifically, we show that the same batch of synthesized GO exhibits (i) potent anticancer activity against MCF-7 breast cancer cells through ROS-mediated apoptosis, mitochondrial dysfunction, and caspase activation, and (ii) remarkable photocatalytic degradation ability toward toxic Rhodamine-B dye under visible light irradiation. This combined biomedical–environmental functionality highlights the multifunctional platform potential of GO, thereby addressing both health and environmental challenges in a single study.

Considering these unique properties, the present study investigates the dual functionality of synthesized GO as both an anticancer agent against MCF-7 breast cancer cells and a photocatalyst for the degradation of Rhodamine-B dye. The synthesized GO nanosheets were systematically characterized by XRD, FTIR, TGA, FE-SEM/EDX, and TEM to confirm their crystallinity, functional groups, thermal stability, and morphology. Their anticancer activity was evaluated through cell viability assays, fluorescence-based ROS detection, and apoptosis-related assays, while photocatalytic efficiency was assessed through dye degradation experiments under visible light irradiation.

Materials and methods

Synthesis of graphene oxide

The preparation of Graphene oxide (GO) was carried out via Modified Hummer’s method, followed by¹². The source materials employed for the preparation of the GO are Graphite flakes, sulphuric acid (H₂SO₄), hydrochloric acid (HCl), hydrogen peroxide (H₂O₂) (30%), and Potassium permanganate (99%). Initially, 95% H₂SO₄ and 5% H₂O were mixed, and later graphite flakes were added. The above mixture was allowed to stir for 30 min. Following the KMnO₄ (18 g), it was added in small portions, and the process was done under ice bath conditions. Once the addition of KMnO₄ was completed, the ice bath was replaced with an oil bath that was maintained at 35 °C overnight. After overnight stirring, 100 ml of water was progressively added, and the mixture was stirred for an additional 30 min, resulting in a change of color from yellow to dark brown. Then the precipitate was extracted from the solution for centrifugation. Further, the precipitate was centrifuged with HCl five times, and then the precipitate was cleaned with distilled water until the pH reached neutrality. Finally, the precipitate was dried in an oven at 70 °C overnight to obtain GO powders.

Characterization of graphene oxide

X-ray diffraction (XRD) using (X-pert PRO diffractometer - Shimadzu, 8201PC) was employed to study the phase purity, structural analysis, and crystallinity of the GO with Cu K_α radiation (λ = 1.54178 Å). The FTIR spectrometer (Quickton, China) was utilized with ATR to quantify the existence of distinct functional groups in the GO. Using a 514 nm laser as the excitation source, a Raman spectrometer (Olympus, Germany) was used to record the Raman spectra of GO. The thermal stability of GO was assessed by thermogravimetric analysis (TA, Q20, USA). A sample of about 8 mg was heated at a rate of 20 °C/min from 30 to 795 °C while being analyzed at 40 mL/min in an air atmosphere. By calculating the first derivatives of the TGA curves as a function of temperature, the DTGA curves were produced. The surface morphology and chemical compositions of GO were measured with a Field Emission Scanning Electron Microscope utilizing a (FESEM, Quanta FEG250, Japan) coupled with energy dispersive X-ray spectroscopy (EDX, Ametax, Z1 analyzer Z230).

Biological properties of GO

Procurement of cancer cells

A freeze condition MCF-7 Human breast cancer cell was purchased from the National Centre for Cell Science, Pune, Maharashtra, India. Dulbecco’s Modified Eagle Medium was used in the entire experiment, 6-well plate, 96-well plate, sodium bicarbonate (0.2%), trypsin, fetal bovine serum albumin (10%), antibiotics of penicillin and streptomycin were received from Sigma Aldrich, Mumbai, India. All the other media, and fluorescent dyes of AO/EB, DAPI, and rhodamine 123 for this work were procured from Merk Scientific & Co, Tamil Nadu, India.

Quantification of cytotoxicity effect using GO

A brief procedure by Thangavel et al.¹³ was followed to detect the cytotoxicity effect of GO. Approximately 1 × 10⁴ cells of monolayer MCF-7 breast cancer cell culture were seeded into a 96-well plate and incubated at 37 °C for 24 h with 5% CO₂. Cancer cells were treated with a series of 25–175 µg/mL concentrations of GO. Subsequently, DMSO was treated in a control culture. After 24 h, 20 µL of MTT solution was carefully mixed into all the GO-treated wells and further maintained for 4 h. After incubation, MTT was aspirated, and 200 µL of DMSO was mixed to dissolve the formazan crystals within the samples. Finally, the absorbance of the culture was read at 590 nm (Measurement) and 600 nm using a microplate reader (Shimadzu, Japan). In addition, DMSO by itself was confirmed to be non-toxic to the cells. Results were measured in triplicate and used to calculate the mean value and standard deviation. The percentage of inhibition was identified using the following formula,

Based on the identification, the half-inhibition concentration of the GO-treated result was used to fix the IC₅₀ concentration of this study. Subsequently, this IC₅₀ concentration value was chosen for further morphological study experiments.

Observation of morphological death using a phase contrast microscope

Initial morphological damages of MCF-7 breast cancer cells after exposure to an IC₅₀ dose of the GO were observed by a phase contrast microscope¹⁴. This morphological damage assay was performed in GO-treated MCF-7 breast cancer cell culture, containing a 6-well plate-soaked cover slip. After exposure, the cover slip culture was allowed to grow at 37 °C for 24 h with 5% CO₂. After, the treated wells were fixed with 4% formaldehyde for 10 min. Afterward, the coverslip was mounted carefully onto the glass slide, and morphologically varied breast cancer cells were observed by a phase contrast microscope at 40×g (Olympus, USA).

Apoptotic cell death of GO by fluorescence microscope

Morphologically damaged MCF-7 breast cancer cells due to exposure to the IC₅₀ concentration of the GO were confirmed by the AO/EB staining method¹⁵. Briefly, 1 × 10⁵ cells were seeded on a coverslip containing a 6-well plate with medium and maintained at 37 °C for 24 h to attach the cells on the coverslips along with the IC₅₀ dose of the GO. After treatment, the cells were harvested and stained with 25 µg/mL of the AO/EB combination and incubated for 10 min. Then, the unbound stains were removed by PBS, and the cells were fixed with methanol and glacial acetic acid for 30 min. Afterward, the nuclear morphology of the cells was observed by fluorescent microscopy and interpreted with control images.

DAPI staining assay

The floating and adherent MCF-7 cells were collected separately after being treated with an IC₅₀ dose of GO for 24 h. Subsequently, the cells were washed with ice-cold 1 × PBS and then fixed with 4% paraformaldehyde for 15 min. Then, the cells were repeatedly thrice using ice-cold 1 × PBS. Afterward, cells were stained with 2 µg/mL DAPI stain and incubated at 37 °C for 45 min. Excess stains were removed by PBS, and fixed cells were observed under a fluorescence microscope at 40× magnification¹².

Internal cell damage by ROS production of GO

The intracellular ROS-producing ability of synthesized GO with their IC₅₀ dose against MCF-7 breast cancer cells was confirmed by a fluorescence microscope using dichlorofluorescein, which is a green color dye used to interact with internal reactive oxygen. Based on the IC₅₀ dose-based cytotoxicity experiment, the culture was stained by DCFH-DA after completing the treatment incubation in a 6-well plate. Then, the excess stains of the cells were washed thrice with PBS and incubated at a 1 h time interval. Subsequently, the ROS-mediated damages of MCF-7 breast cancer cells were observed under a fluorescence microscope. The control and treated images of the ROS-based cells were interpreted to confirm the GO efficiency against MCF-7 breast cancer cells¹⁶.

Detection of mitochondrial membrane potential (Δψm) of GO

The mitochondrial membrane potential (Δψm) of the MCF-7 breast cancer cells was determined by the fluorescent dye rhodamine 123¹⁷. Here, the cytotoxicity procedure was performed on a range of MCF-7 breast cancer culture quantities (5 × 10³ cells per well) treated with an IC₅₀ dose of GO in a 6-well plate, using DMEM medium, and allowed to incubate at 37 °C for 24 h. The next day, the cells were taken out after washing the culture twice. Then, the culture was stained with 5 µg/mL of rhodamine 123 in PBS and maintained at 37 °C for 15 min in the dark. Both the treated and untreated cells were observed under an inverted fluorescence microscope. The fluorescence intensity was calculated using a Microplate Fluorescence Reader (FLx800, BioTek). In living cells, Rhodamine 123 can penetrate through the plasma membrane and is sequestered by mitochondria.

Enzymatic activity of GO in caspase

The protease activity of caspase-3 and caspase-9 of MCF-7 breast cancer cells was quantified by colorimetric assay with the modified procedure of Doghish et al.¹⁸. Approximately 2 × 10⁴ well-matured MCF-7 breast cancer cells were aliquoted in fresh media containing a 6-well plate along with an IC₅₀ dose of the GO for treatment and incubated the plates at 37 °C for 24 h. Cisplatin served as an internal control. After 24 h, all the cells of the 6-well plates were centrifuged at 10,000 rpm for 15 min and then washed with 10 PBS. Afterward, cells were lysed with 100 µL cold lysis buffer and incubated on ice for 45 min. After lysing, the cells were centrifuged again with the same parameters, and the supernatant was collected carefully without any debris. The samples were shifted to fresh tubes, and the protein concentration of treated and untreated samples was monitored by the Bradford method. Subsequently, 100 µL supernatant having 250 µg of protein-containing samples was diluted with 2 × 10 reaction buffer (100 µL). Then, caspase substrate was added carefully, and the samples were incubated at 37 °C for 4 h in dark conditions. Finally, the functionalized samples were quantified at 450 nm at a microplate reader (Quanta Microplate reader, FEI, Micro, UK).

Photocatalytic degradation of rhodamine-B

A UV–visible spectrophotometer (Perkin Elmer, Lambda 11) was used to measure the photocatalytic activity of GO NSs against Rh-B dye molecules. Each experiment involved mixing approximately 50 mg of manufactured photocatalyst samples with 100 mL of Rh-B aqueous solutions (1 × 10⁻⁵ M), magnetically spinning the suspension for 30 min in the dark, and then exposing the solution to visible light radiation at 20 min intervals. The photocatalytic degradation process was tracked by measuring the intensity fluctuations of absorption peaks in the visible light spectrum at a wavelength of 550 nm, which corresponds to the maximum absorption peaks of Rh-B. Each test was carried out in triplicate.

Statistical analysis

Data analysis and graphical representations were performed using OriginPro 9.0 (OriginLab Corporation, Northampton, MA, USA; https://www.originlab.com) and GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA; https://www.graphpad.com). The entire study results data are maintained as mean ± standard error of the mean. The one-way ANOVA test was performed as a Dunnett’s multiple comparison method for comparing the control groups. Calibration curves for Rh-B dye molecule degradation were constructed using simple linear regression analysis. Data are mean ± standard error of the mean and n = 3. The P-values < 0.05 of the result were indicated as statistically significant.

Result and discussion

Characterization of GO

The XRD analysis reveals the phase purity, structural analysis, and crystallinity behavior of the synthesized graphene oxide. Figure 1a displays the XRD pattern of GO, which exhibits crystalline behaviour. The XRD peaks were observed at 10.3° and 42.5° for the synthesized GO, which well matched the existing literature^19,20. The additional peaks were observed at 2θ = 21.4° and 26.6°, suggesting that the GO structure is not fully oxidized and retains partially interconnected graphitic domains^21–23.

Fig. 1.

Synthesis and characterization of graphene oxide (GO): (a) XRD pattern, (b) FTIR spectrum, (c) Raman spectrum, (d) and TGA with DTGA traces.

The presence of various functional groups attached to GO is detailed using FTIR analysis as displayed in Fig. 1b. The O–H stretching vibration observed at 3518 cm⁻¹ belongs to the water molecules present within the structure. Symmetric stretching vibration of the C–H bond (2805 cm⁻¹) and the asymmetric stretching vibrations of the C–H bond (2907 cm⁻¹). The carbonyl C–O stretching vibration was observed at 1720 cm⁻¹. The CO₂ and C=C and stretching vibration at 2391 and 1752 cm⁻¹. The C=O stretching vibration, characteristic of sp² hybridization, is observed at 1561 cm⁻¹. The C–O–C stretching of the epoxy group was found at 1105 cm⁻¹. Hence, the existence of different oxygen-containing functional groups, such as hydroxyl, epoxy, carboxyl, and carbonyl, is present within the GO structure, as confirmed by the FTIR analysis. The FTIR study conducted by Neeru et al.^24,25, Nathir et al.²⁶, is in excellent agreement with the present work.

The structural characteristics of GO were further analysed using Raman spectroscopy (Fig. 1c). The Raman spectrum of GO has two bands identified at 1352 cm⁻¹ (D band) and 1592 cm⁻¹ (G band). The D band at 1352 cm⁻¹ with A_1g mode corresponds to disorder caused by the disorder in the structure, dangling sp² carbon bonds, and edge effect, and graphite lattice vibrations, whereas the G band at 1576 cm⁻¹ represents E_2g mode having plane stretching motion between sp² carbon atoms. Similar bands observed by Raman spectra conducted by Chadha et al.²⁷, Yoo et al.²⁸, Sánchez-Campos et al.²⁹ agreed well with the current work.

Figure 1d illustrates the TGA/DTGA analysis of graphene oxide (GO), which provides insights into its thermal stability. An initial weight loss of about 8.4% below 100 °C corresponds to the evaporation of physically adsorbed water molecules. A further weight loss of 14.1% from 198 °C is attributed to the removal of intercalated or adsorbed water. The prominent endothermic peak in the DTGA curve around 219 °C indicates the thermal decomposition of oxygen-containing functional groups. A major weight loss of approximately 25.8% above 618 °C is associated with the decomposition of labile oxygenated groups, such as hydroxyl, epoxy, carboxyl, and alkoxy functionalities. At higher temperatures, a smaller weight loss of about 3.16% corresponds to the gradual degradation of the carbon backbone. These findings are consistent with earlier reports by Wojtoniszak et al.³⁰ and Yap et al.³¹, further confirming the characteristic thermal behavior of graphene oxide.

The surface morphology of the synthesized GO was analyzed by FESEM, which revealed thin, overlapping, and aggregated sheet-like structures (Fig. 2a,b), consistent with previous reports³². TEM further confirmed the sheet-like morphology with smooth surfaces and few stacked regions, indicating the high transparency of GO (Fig. 2c,d). The EDX spectrum (Fig. 2e) showed only carbon and oxygen, with atomic percentages of 65.55% and 34.45%, respectively, confirming the successful synthesis of pure GO by a simple and cost-effective method.

Fig. 2.

Surface morphology of the GO using FE-SEM. (a, b) low and high magnifications of GO structural morphology by FE-SEM, and (c, d) observation of GO morphology by TEM analysis. (e) Available elemental composition of the synthesized GO by EDX spectrum.

Quantification of the cytotoxicity effect of GO

The MTT assay result of GO-treated cytotoxicity effect in MCF-7 breast cancer cells was indicated in Fig. 3. Based on the observation, the cytotoxicity effect was increased at various concentrations, and confirmed the result depends in a concentration-dependent manner. The result exhibited with decreased cell quantity and increased cytotoxicity was observed after treatment with a 175 µg/mL concentration of GO. The result of the cytotoxicity assay confirmed that the interaction of GO against MCF-7 breast cancer cells was very effective, and it was directly proportional to the concentration of GO. As a result, inhibition was started at 25 µg/mL concentration, and substantially increased inhibition, as well as half inhibition range, was shown at increasing concentration of 175 µg/mL (Fig. 3a). The result suggested that the GO has increased the anti-cancer property at increasing concentration as well as concentration-dependent mode. At a concentration of 175 µg/mL, 52% of the cells were dead in triplicate mode, and it was fixed as an IC₅₀ dose for this research. The present work was compared to prior research work on the cytotoxicity effect against MCF-7 breast cancer cells, the inhibition was concentration-dependent³³. Previous reports of concentration dependent anti-cancer effect of GO against various cancer cells, as evident in the Table 1.

Fig. 3.

Detection of cytotoxicity effect and morphological differentiation of GO against monolayer MCF-7 breast cancer cells seeded 96-well plate and incubated at 37 °C for 24 h with 5% CO₂: (a) Detection of IC₅₀ dose of GO against MCF-7 breast cancer cells by spectrophotometer analysis. (b) Morphological differentiation of control and (c, d) GO treated irregular morphology of MCF-7 breast cancer cells by phase contrast microscope. Data are mean ± standard error of means and n = 3 for each group. ^**p < 0.001; ^****p < 0.0001 compared to the vehicle control group, ns, not significant.

Table 1.

Anti-cancer activity of GO and its composites against MCF-7 breast cancer cells at concentration-dependent inhibition.

Material	Cell line	Concentration (µg/mL)	References
GO	MCF-7 Breast cancer cells	250	Motafeghi et al.37
GO	MCF-7 Breast cancer cells	800	Zuchowska et al.38
rGO	MDA-MB 231 Breast cancer cells	300	Krętowski et al.39
GO	FM3A-Luc Breast cancer cells	500	Thabang et al.40
GO	FM3A-Luc, OV 2944-HM-1, NFSAY83 and KM-Luc/GFP	500	Thabang et al.40
GO	COLO 205 and 293T cells	200	Ibrahim et al.41
GO-DSPE-PEG-Tf	MCF, MDA-MB 231 and SK-BR-3	250	Johnson et al.42
Gr/Ni	LNCaP prostate cancer cells	300	Rajivgandhi et al.44
Fe3O4/rGO	MCF-7 Breast cancer and A549 lung cancer cells	200	Karthika et al.43
GO	MCF-7 Breast cancer cells	175	Current study

GO; Graphene oxide, rGO; Reduced graphene oxide, GO-DSPE-PEG-Tf; GO nanoribbons conjugated with 1, 2-distearoyl-sn-glycero-3 phosphoethanolamine-poly (ethylene glycol)-transferrin, Gr/Ni; Graphene/Nickel, Fe₃O₄/rGO; Iron oxide/reduced graphene oxide.

Similarly, the GO is the most effective 2D nanomaterial, which is frequently used against various cancer cell lines, and it can destroy various cancer cells³⁴. Recently, the compromised MCF-7 breast cancer cells due to oxidative stress after exposure to GO were reported by Mahesh et al.³⁵. Similarly, the concentration-dependent GO was very effective against MCF-breast cancer cells, and it completely eradicated them, which was confirmed by MTT assay^14,36. Notably, the GO has anticancer ability against various cancers cells, and its proliferation efficiency is performed to produce the prooxidant properties, which produced a significant quantity of reactive oxygen species, nuclear damages, cell cycle arrest, oxidative stress-mediated nucleus damages, and intracellular modification, thereby resulting in a substantial decrease in the viability of MCF-7 breast cancer cells¹.

Further, the decreased cell quantity and differential morphological observation of MCF-7 breast cancer cells were shown in their external appearance due to the influence of 175 µg/mL of GO, and indicated in the phase contrast microscope (Fig. 3c). After completing the cytotoxicity assay, the damaged and control MCF-7 breast cancer cells of phase contrast microscope images were interpreted, and suggesting that the MCF-7 breast cancer cells were more compromised to IC₅₀ value of GO. Compared with the untreated control, the cell quantity and irregular morphology were observed in the GO-treated images (Fig. 3d). Whereas, tightly intact adherent cells with original spherical morphology of control MCF-7 breast cancer cells were shown in Fig. 3b. In addition, a more sparse cell with compromised structure of detachment as well as significant decrease of MCF-7 breast cancer cells were differentiated from original cells (Fig. 3c,d). All the primary observation results indicated that the GO is an effective anti-cancer material. In addition, clear supporting evidence was reported by Karina de Oliveira et al.⁴⁵, who reported that the original spherical shape of the MCF-7 breast cancer cells was altered due to the induction of nanoparticles. Different nanoparticles have different biological properties against cancer cells based on the concentration-dependent mode⁴⁶.

Apoptotic cell death of GO by fluorescence microscope

Graphene oxide is an excellent 2D-based carbon material that provides an excellent anticancer platform and an excellent biocompatible agent compared to any other nanomaterial⁴⁷. Apoptosis is a complex process that is used to detect cancer cell death due to the influence of nanomaterials in various cellular signalling proteins^48,49. The decreased cancer cells and differentiation of live/dead cells nature due to the apoptogenic activity of 175 µg/mL of GO after being interpreted with control cells were depicted in Fig. 4. To detect the apoptotic cells, the dual stain of AO/EB fluorescence dye proliferated in the damaged cell organelles and scattering with red color appearance, suggesting apoptosis-related modification happened in 175 µg/mL of GO-treated MCF-7 breast cancer cell growth and quantity of cancer cell viability, necrotic cell growth and apoptotic cells in both control and treated GO against MCF-7 breast cancer cells were clearly shown in Fig. 4. Figure 4a indicated that the clear morphology of untreated cancer cells was suggested that the cells did not affect any harmful effect, which exhibited as matured growth cells. Whereas, the GO-treated breast cancer cells showed more dead cells, which exhibited differential morphology with abnormal red color cells (Fig. 4b). In addition, significant alterations, including crushed morphology, inconsistent growth, cleavage of the cytoplasmic membrane, and other internal organelle damage showed green/red colors in Fig. 4c. As per the mechanism, AO is a green fluorescence dye that can penetrate the intercellular membranes of both live/dead cells, whereas EB is another fluorescence dye that can enter damaged membranes only due to the more cationic charges¹⁴. After AO penetration, the damaged cancer cell membrane was intercalated with DNA molecules, which could be exhibited as fluorescence-scattered green color images. On the contrary, EtBr could enter into the damaged cancer cell membrane, dead cells, and also late apoptotic cells, which could be exhibited as fluorescence-scattered red color images¹⁵. Due to the influence of GO in MCF-7 breast cancer cells, the cells underwent a decline phase, and more necrotic cells were formed, which emitted orange-red fluorescence. Based on the given result of Fig. 4, the exhibited fluorescence coloring pattern of cancer cells differentiated the necrotic and condensed cells due to the administration of GO. More precisely, the green color transitions to an orange tint alteration can be confirmed by the initial apoptosis and the frequent compaction of cell nuclei. Based on this mechanism, the exhibited result of GO-treated MCF-7 breast cancer cells indicates that the early apoptosis and late apoptotic cells were emitted with bright green fluorescence and red-orange fluorescence images. The compromised MCF-7 breast cancer cells indicated that the damaged, condensed, and fragmented chromatin was formed due to the induction of GO^34,50. The more apoptotic cells with disparities in the outer morphology of the red color emission of the fluorescence images indicated that the MCF-7 breast cancer cells, suggesting compromised GO. Also, more apoptotic cells with nuclear shrinkage and condensed, fragmented morphology were clearly shown in red (Fig. 4c) and late apoptosis and nuclear condensation of the treated cells showed with orange color (Fig. 4d). As per the AO/EB dual staining theory, the high intensity of the green color fluorescence appearance indicates that the MCF-7 breast cancer cells formed well-structured patterns with live cells. Whereas the high-intensity emission of the red color appearance within GO-treated cells images was suggested, the cells were compromised by GO and exhibited both light green and bright red, along with orange color^48,49. In addition, the cells were shown with damaged and fragmented DNA due to nuclear shrinkage and blebbing, suggesting that apoptosis occurred due to the exposure to GO. Furthermore, the bright green color appearance due to the intercalation of AO indicated that the damages were shown after the cells were in the early apoptosis stage. As well as the high number of red color and orange color cells suggested that the intercalation of AO and EB happened in the damaged or fragmented cells in the late apoptotic process (Fig. 4d). Finally, the dual staining assay confirmed that the damage of DNA, nucleus, and more apoptotic bodies of the MCF-7 breast cancer cells were compromised due to the IC₅₀ dose GO, which could predominantly induce the apoptosis in MCF-7 breast cancer cells. The morphological damages of MCF-7 breast cancer cells through enhanced ROS production by IC₅₀ concentration of GO, as reported by Samson et al.^10,51. The high fluorescence intensity of EB into GO-treated MCF-7 breast cancer cells was effectively observed by AO/EB staining assay, and suggested that the cancer cell morphology was damaged through ROS produced by GO, and confirmed as an effective anti-cancer agent against breast cancer cells⁹.

Fig. 4.

Compromised intracellular morphology of MCF-7 breast cancer cells after treatment with an IC₅₀ concentration of GO. (a) The AO/EB staining of untreated control exhibited green color morphology, (b) inhibition of cancer cells exhibited red/orange color, (c) early and (d) late apoptotic cell death indicated damaged structure of MCF-7 breast cancer cells after treatment with GO by fluorescence microscopy analysis. (e) control, (f) IC₅₀ concentration of GO-treated cells exhibited early apoptotic cell death with a damaged nucleus of bright blue color, and (g) late apoptosis of MCF-7 breast cancer cells with increased necrotic cells with bright white color were shown after staining with DAPI.

Detection of nuclear damage of GO by DAPI

The utilization of DAPI staining is the confirmation method for the detection of ROS-mediated nuclear damage in cancer cells⁵². In this current study, the cluster of nuclear content leakage shrinkages of treated cells with chromatin condensation was exhibited. The nuclear and cytoplasmic leakages of the result suggested that the genetic material and fragmented nuclei within the cancer cells were compromised due to the exposure of IC₅₀ of GO. The DAPI dye is the specific dye that can be found inside the nucleus of cancer cells⁵³. In our result, the sharp edge of the GO interacted with the negative charges of the outer nuclear membrane through electrostatic interaction. Subsequently, the excess production of hydroxyl radicals induced the oxidoreductase pathway, and it triggered apoptosis consistently. The differentiation of nuclear damages of 175 µg/mL of GO-treated (Fig. 4f,g) and matured nucleolus of untreated (Fig. 4e) GO of MCF-7 breast cancer cells was observed. The bright white color appearance of the GO-treated image suggested, that the damages occurred due to the oxidative stress interaction in the outer cell membrane of MCF-7 breast cancer cells (Fig. 4g). The supported result was published by Asha et al.,⁵⁴ the internal nucleus damages of MCF-7 cancer cells were confirmed by DAPI stain at their IC₅₀ dose treatment of 175 µg/mL. Similarly, graphene is an advanced nanomaterial having excellent anti-cancer properties against cancer cells, especially nuclear damage of MCF-7 breast cancer cells⁵⁵. Based on the above-mentioned previous research evidence, the current result suggests that the MCF-7 breast cancer cells were compromised to the IC₅₀ dose of tested GO, and confirms the nuclear membrane damage with more abnormalized apoptotic cells. Further, the more intensified fluorescence observation with the bright appearance of the MCF-7 breast cancer cells was viewed in GO-treated cells using a fluorescence microscope.

Internal cell damage by ROS production of GO

The fluorescence microscopic images observed in Fig. 5b,c, suggest a direct interaction between the IC₅₀ concentration of GO, which exhibited overproduction of intracellular ROS levels. In this process, the free radicals of the sharp-edged GO are the ROS factory against MCF-7 breast cancer cells, indicating more dead cells in 175 µg/mL of IC₅₀ dose-treated cells. To detect ROS-mediated intracellular damage in MCF-7 breast cancer cells, a specific DCFH-DA dye was used. Specifically, DCFH-DA dye was used to determine the amount of ROS based on the intensity of the green fluorescence emission. In control, the original morphology of the MCF-7 breast cancer cells with bright green color fluorescence morphology was observed (Fig. 5a). Whereas, the strong fluorescence of bright green color appearance with some chemical forces on the membrane cells organelles of the GO was shown in treated cells (Fig. 5b,c).

Fig. 5.

Intracellular ROS production after exposure of GO against MCF-7 breast cancer cells at IC₅₀ concentration: (a) Matured growth of MCF-7 breast morphology with the normal green color of untreated, (b) intracellular ROS-mediated damages, (c) increased apoptotic and necrotic cells of the MCF-7 breast cancer cells morphology after treatment with GO by fluorescence microscope. (d) Quantification of ROS production after treatment with GO against MCF-7 breast cancer cells by spectrophotometer. Data are mean ± standard error of means and n = 3 for each group. ^****p < 0.0001 compared to the vehicle control group.

Parallelly, ROS-mediated MCF-7 breast cancer cell inhibition was quantitatively measured after treatment with GO, and results indicated there is significant inhibition at increasing concentration. In control cells, the mature growth of the cancer cells with no alteration was observed. In the case of GO-treated culture, the cell growth was gradually decreased through the generation of ROS, and it entirely disrupted the cancer cells quantity at 175 µg/mL. A report on MiO theory, which states that the generation of ROS based on different concentrations has significant permeable behaviour in intracellular cancer cells, which leads to damage to the entire cancer cell parts, including mitochondria, nucleus, and DNA . Based on the liquid media dispersion effect, the GO produced more ROS, and it damaged the internal membrane of the MCF-7 cancer cells through a frequent apoptosis process. After careful staining with DCFH-DA, the IC₅₀ concentration of GO-treated cultures was quantitatively measured by a UV-spectrophotometer, and the result of inhibition was calculated in percentages. The ROS-based cell death quantity after being compared with doxorubicin, an anti-cancer drug, was discussed in Fig. 5d. In both, 50 µg/mL, 100 µg/mL, 150 µg/mL, and 175 µg/mL concentrations were performed randomly based on the MTT assay result, and it indicated that the 175 µg/mL concentration of GO produced more cell death after ROS generation than doxorubicin. The result confirmed that the GO stimulated the ROS in the intracellular membrane surface of cancer cells, causing more oxidative stress-based damage and more dead cells. Subsequently, internal active parts of DNA, proteins, amino acids, chromatin, mitochondria, nucleus, and other organelles were destroyed. In this stage, the cells underwent late apoptosis, creating more damage in the nucleus and chromatin region³⁴. In addition, the excess accumulation of ROS in intracellular membrane surfaces stimulates up- and down-regulating apoptotic pathways in MCF-7 breast cancer cells, which destroys entire cancer cells³⁵. Evidently, continuous reduction of m due to the effect of overproduction of ROS by IC₅₀ of GO suggested that more radicals were attacked on the membrane phospholipids of the cancer cells⁵². Recently, Hendry Moses et al.⁵⁶ demonstrated that the fluorescence intensity of the GO dosage against MCF-7 breast cancer cells due to the ROS levels showed that the GO is an effective anti-cancer agent. An increasing concentration of GO showed a significant increase in intracellular levels of ROS compared to the control^33,57. Based on the previous study result, our results indicated that the increasing concentration of GO produced an excess level of ROS against cancer cells, and it reduced the survival rate in cancer cells. Further, the ROS induces programmed cell death resulting from cellular damage of MCF-7 breast cancer cells. The noteworthy report of more apoptosis occurred due to the interaction of hydroxy radicals from GO to cancer cells through phospholipid peroxidation, as documented by Sakthivel et al.¹⁷. Furthermore, the GO could trigger caspase-3, which is a crucial enzyme in the process of programmed cell death. It is noteworthy that there was also an increase in apoptosis observed in MCF-7 breast cancer cells. Here, the evidence of ROS-damaged MCF-7 breast cancer cells due to the exposure to GO indicated that the cells underwent alterations that resembled the characteristic hallmarks of apoptosis. The detected alteration of the result showed more apoptosis in the decline phase of dead cells.

Detection of mitochondrial membrane potential (Δψm) of GO

In cancer cell eradication, the inhibition of the mitochondrial membrane is an important factor to observe the intracellular damage of the cancer cells or not⁵⁸. In this research, the proliferation efficiency of GO with the loss of Δψ_m on the sensitized outer layer of the mitochondrial membrane is the initial step of apoptosis. After treatment with silver nanoparticles, the lipophilic cationic Rhodamine-123 dye was used to detect the accumulation of Δψ_m loss on the inner mitochondrial membrane of mCF-7 breast cancer cells^50,59. Likewise, the depolarization of the mitochondrial membrane of MCF-7 cells after exposure to GO was examined under a fluorescence microscope using Rhodamine 123, and it was found that the fluorescence intensity of Rhodamine 123 was decreased, which suggested that the level of Δψm was also decreased significantly (Fig. 7d). At the IC₅₀ dose of GO, the cellular uptake of fluorochrome was reduced in treated cells when compared to untreated MCF-7 breast cancer cells. Based on this observation, the current evidence indicates that the intensification of fluorescence was very high in untreated cells (Fig. 7a), whereas the fluorescent signal significantly reduced after treatment with an IC₅₀ of 175 µg/mL GO (Fig. 7b,c). Hence, the result suggested that the IC₅₀ concentration of GO substantially increased the apoptotic cells and significantly decreased the Δψm level due to the enhancement of ROS. In particular, the significant reduction of Δψm in MCF-7 breast cancer cells due to the increased apoptosis by GO. Based on the mechanism, the result suggested that the mitochondrial membrane damage occurred due to the loss of Δψm, which leads to bulk cell death, supported by Hashemi et al.^60,61. Finally, the current study suggests that the synthesized GO effectively depolarized the membrane potential in mitochondria and enhanced the overproduction of ROS, suggesting that the GO is an excellent anti-cancer material against MCF-7 breast cancer cells. The Fig. 6 explains the step-by-step procedure of the synthesis of modified Hummer’s to the structural/chemical features of GO as well as their complete intracellular mechanism in the MCF breast cancer cells.

Fig. 7.

Mitochondrial damages and up-regulating caspases activity of MCF-7 breast cancer cells through apoptosis process: (a) untreated control cells with green color matured cells, (b, c) mitochondrial damages of red color cells, (d) Quantitative measurement of fluorescent intensity on mitochondrial membrane, and (e) Up-regulating caspases enzyme activity at IC₅₀ concentration of GO NSs treated. Data are mean ± standard error of means and n = 3 for each group. ***p < 0.0005 compared to the vehicle control group.

Fig. 6.

Schematic diagram that links the modified Hummer’s synthesis steps to the structural/chemical features of GO, and then to the cellular mechanism.

Upregulating the enzymatic pathway of GO

Caspases are the essential enzymes present in cancer cells, which are used to trigger apoptosis, cell death, and invasion into the cancer cells. Particularly, upregulation of Caspase-3 and caspase-9 is the prime factor to enhance the ROS-mediated apoptosis in cancer cells, as well as the influence of various nanomaterials to enhance the expression of caspases in cancer cells, which leads to enhanced continuous apoptosis, is an interesting approach to eradicate the cancer cells⁵¹. In addition, expression of caspases helped to enhance the ROS-mediated apoptotic cell death at the intracellular level, and also triggered the apoptosis against the mitochondrial region of cancer cells, which leads to the decline phase⁴². Supportively, our previously reported evidence of the modified Hummers method of synthesizing GO enhanced the caspases in A549 lung cancer cells, and increased the apoptotic cell death in a concentration-dependent manner⁶². At 175 µg/mL concentration, a continuous decrease of MCF-7 breast cancer cells due to the activation of the signalling cascade in caspase enzymes was observed after the generation of ROS-mediated apoptosis. Based on the understanding of concentration-dependent inhibition, the caspases-3 and caspases-9 enzymes were activated, leading to an increase in the upregulating pathways in MCF-7 breast cancer cells (Fig. 8). The colorimetric results suggested that more precipitation was observed at 175 µg/mL treated cells than in the internal control of doxorubicin. After interpretation, GO-treated cells were more compromised than doxorubicin-treated cells due to the effect of the highest apoptosis, which was enhanced by ROS production. In addition, GO stimulated caspase enzymes twice as much as the control drug of doxorubicin at the IC₅₀ concentration. When GO concentration was increased, the up-regulation of caspase pathways was stimulated by apoptosis-mediated cell death, and cells underwent a decline phase. At this point, the maturation of cells was arrested, and their intracellular granule production indicated the failure of growth cells as well as the increase in precipitation. In control, the maturation of cells and G₁–G₂ phases were optimized, and the high-level quantity of the cells was measured. Among the results, the current study of synthesized GO confirms that the upregulation of caspase enzyme activity increased over time compared to control doxorubicin due to the increasing concentration of GO. Here, Fig. 7e confirms that GO has a more efficient ability to enhance the upregulating pathway of caspase-3 and caspase-9 to damage the mCF-7 cancer cells through excess ROS production.

Fig. 8.

The graphical representation showed the mechanism by which GO induced apoptosis through the ROS-mediated mitochondrial pathway.

Specifically, in our study, the observed ROS-based damage is predominantly associated with intrinsic ROS generation. This is initiated within the mitochondria, where exposure to GO disrupts the mitochondrial membrane potential⁶³. The loss of mitochondrial membrane integrity facilitates leakage of electrons from the electron transport chain, leading to the overproduction of superoxide anions (O₂^·−) and hydroxyl radicals (·OH)⁶⁴. These elevated ROS levels induce oxidative stress, resulting in mitochondrial dysfunction, lipid peroxidation, and damage to mitochondrial DNA⁶⁵. Importantly, the collapse of the mitochondrial membrane potential promotes the release of cytochrome c into the cytosol, which subsequently activates caspase-9 and caspase-3, driving intrinsic apoptotic signalling pathways⁶⁶. The morphological alterations observed in our fluorescence staining (AO/EB, DCFH-DA, and Rhodamine 123 assays) further support this mechanism, as they reveal nuclear condensation, membrane blebbing, and apoptotic body formation consistent with mitochondria-mediated cell death. The Fig. 8 explains the mechanism of up-regulation of apoptotic genes and down-regulation of anti-apoptotic genes due to the cytochrome C production on the mitochondrial membrane by GO.

Photocatalytic degradation of Rhodamine-B

By tracking the degree of Rh-B dye molecule degradation using UV–visible spectrophotometry, as illustrated in Fig. 9, the visible light photocatalytic degradation capability of GO was investigated. When exposed to visible light at a wavelength of 550 nm, 46.9% of the Rh-B dye molecules broke down in 120 min for GO (Fig. 9a). The time dependence of Rh-B concentration variations (C/C0) upon exposure to visible light at various time intervals for GO photocatalysts is displayed in Fig. 9b. The effectiveness of the produced GO NSs in breaking down Rh-B dye molecules was assessed under the same experimental conditions as a function of irradiation time. 44.9% of the Rh-B dye molecules were eliminated by GO NSs following 120 min of visible light degradation (Fig. 9c).

Fig. 9.

(a) Graphene sheet-based photocatalytic degradation against Rh-B is shown schematically. (b) Rh-B containing solutions treated with graphene nanosheets and their time-dependent UV–visible absorption spectra; (c) Rh-B exposed to visible light while graphene nanosheets were present; and (d) Photocatalytic degradation (%) vs. time using graphene nanosheet calibration plots.

According to Selvam et al.^67,68, the inclusion of GO NSs may have contributed to this unusual outcome by reducing electron-hole pair recombination, improving light-harvesting capacity, and dramatically raising the catalyst’s photocatalytic activity. Here, Fig. 9c,d show how Rh-B dye molecules are photocatalytically irradiated when exposed to light in the presence of GO NSs. With R² values of 0.995, linear correlation trends of Rh-B degradation using GO NSs were discovered (Fig. 9c). The suggested processes explain how GO NSs photocatalytically break down Rh-B dye molecules when exposed to visible light. According to Thakur et al.^69,70, GO NSs’ wide surface area allows them to effectively adsorb dye molecules on the photocatalyst surface and generate π – π interactions between dye molecules. Nanomaterials may increase the production of oxy radicals from interactions with oxygen molecules and cause dye molecules to oxidize by limiting the recombination of excited electrons in the CB with holes in the VB^71,72. The photodegradation of dye compounds by GO NSs is initiated by oxidative and reductive reactions⁷³.

Conclusion

Nanotechnology continues to provide transformative opportunities in biomedical and environmental applications, with 2D graphene-based materials standing out due to their unique physicochemical properties. In the present study, GO demonstrated significant anticancer potential against MCF-7 breast cancer cells, a widely used and clinically relevant model for studying enzyme-dependent breast cancer. The ROS-mediated apoptosis pathway observed in MCF-7 cells confirmed that GO effectively induced mitochondrial dysfunction, cytochrome c release, and increased apoptotic signaling, ultimately leading to dose-dependent cancer cell death. This highlights the potential of GO as a novel therapeutic platform for breast cancer treatment with reduced side effects compared to conventional therapies. In parallel, the photocatalytic activity of the synthesized GO was validated through visible light-driven degradation of Rh-B dye. The enhanced photocatalytic efficiency was attributed to oxygen vacancies and localized energy states within GO, which minimized electron–hole recombination and promoted reactive oxygen species generation. The successful degradation of Rhodamine-B not only demonstrates the multifunctionality of GO but also underscores its potential for environmental remediation of toxic dye effluents. Overall, the dual functionality of anticancer activity against breast cancer cells and efficient photocatalytic degradation of organic pollutants indicates, GO is a promising nanomaterial for both biomedical applications and sustainable environmental technologies.

Author contributions

Chenthis Kanisha Chelliah – Designed the concept, conducted the investigation, and wrote the original manuscript. Govindan Rajivgandhi – Contributed to the anti-cancer activity. Maruthupandy Muchuchamy, Gnanasekaran Chackaravarthi, Govindan Ramachandran - Investigation of anti-cancer work, interpretation, data curing and review & editing, language correction, grammatical mistakes, typo errors of the Manuscript. Liu Yong-Hong , Osama Abdalla Abdelshafy Mohamad , Lee Sui, and Wen-Jun Li – Participated in the synthesis & characterization of GO. Vijayabhaskara Rao Bhaviripudi- Contributed to the characterization of TGA, formal analysis, data curation, and anti-cancer activities of the manuscript. S. Sathik Basha – Supervisor, contributed to the characterization of TGA, Formal analysis, Data curation, and anti-cancer activities of the manuscript. Kiran Shahapurkar -– Contributed to the photocatalytic study of the manuscript. Gezahgn Gebremaryam – Supervisor, contributed to the entire manuscript and interpretation of all the GO characterization.

Data availability

Data will be made available from **Chenthis Kanisha Chelliah** on a reasonable request.

Declarations

Competing interests

The authors declare no competing interests.