Graphene oxide–microplastic hybrid showcase elicited discrepancy through intrinsic interaction mediated steatosis, and apoptosis in macrophages
Adrija Sinha, Arghyadeep Mayur, Snehasmita Jena, Aishee Ghosh, Mrutyunjay Suar, Suresh K. Verma
TL;DR
This study shows that a hybrid of graphene oxide and microplastics causes more cell damage than either alone, highlighting risks from their environmental interactions.
Contribution
The study reveals amplified toxicity of a graphene oxide–microplastic hybrid through combined experimental and computational analysis.
Findings
GO@MP hybrid caused higher lipid peroxidation and mitochondrial damage than individual GO or MP.
GO@MP interactions with proteins like PEX5, BCL2, and Caspase 3 led to steatosis and apoptosis in macrophages.
Atomic-level modeling showed structural and functional disruptions in proteins due to hybrid interactions.
Abstract
The widespread natural abundance of microplastics (MP) has been recognized to pose significant global health concerns, particularly due to limited understanding of their biological interactions. With the uncontrolled increase in MP accumulation in the environment, their interaction with xenobiotics like nanomaterials used for different biomedical and environmental applications is likely to be enhanced, raising concern over the advanced toxicological impacts. Hence, it is important to deduce their threatening toxicity to the biological niche, including humans. This study deduces the cytotoxicity of a green-synthesized GO@MP hybrid using macrophage cells, integrating experimental and computational methods. Physicochemical characterization was performed using FTIR, SEM, and DLS. Toxicological assessment revealed that GO@MP significantly reduced cell viability, primarily via surface…
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Taxonomy
TopicsGraphene and Nanomaterials Applications · Nanoparticles: synthesis and applications · Phagocytosis and Immune Regulation
Introduction
1
Nanotechnology has sprung up as a potent tool for tackling global issues and promoting sustainable development. Researchers have unveiled new possibilities for the application of nanomaterials in several sectors, including energy, healthcare, agriculture, construction, and environmental conservation [1]. Several metal and metal oxide nanoparticles have shown promising potential to be utilized in different biomedical sectors, including bio-imaging, scaffolds and implants, targeted pharmaceutical goods, and novel diagnostic instruments [2]. Interestingly, Graphene oxide nanoparticles have gained popularity in leaps and bounds because of their strong adsorption capacity and large surface area. It is versatile for functionalization in a variety of fields, specifically in drug and gene delivery [3], biosensing [4], bioimaging and scaffold preparation [5] due to its distinctive atomic and molecular characteristics. Structurally, it can be recognized as a derivative of graphene, a carbon-based nanomaterial, consisting of a monolayer of sp^2^-hybridized carbon atoms organized in a hexagonal honeycomb lattice. This configuration provides unique physiochemical characteristics that not only serve multiple purposes but also improve the capacity of Graphene Oxide to combine with many other nanomaterials and chemicals to form hybrid materials conveniently [6,7]. Along with the advancements in industrial and lab-scale research and development, its after-usage discarding into the environment has also expanded. Thus, concerns have been raised regarding its toxicological effects on human health and the eco-biological niche [8]. Attempts have been made to investigate the toxicological effects of Graphene oxide and its derivatives using different in vitro and in vivo models. The interaction between Graphene and many cell types has been scrutinized, however, validated results reported on its impact on safety, immune cell function, and cell viability are lacking. For instance, certain kinds of Graphene structures are linked to toxicity in growing cells and autophagy induction, while some literature explains how Graphene plays a beneficial role in wound healing and anti-inflammatory responses [9]. The above-stated facts claim that accumulated post-use Graphene oxide in the environment can combine with other xenobiotics and new pollutants to create hybrids that can be toxic at the biological level in either antagonistic or synergistic ways. The fact needs a thorough investigation for both ecological and biomedical concerns.
On the other hand, plastic being abundant in the environment due to its far-reaching social benefits has become the subject of public concern in recent years. Microplastic pollution has drastically intensified with time, given that it not only acts as a toxic pollutant but also can carry additional contaminants of concern and transport them through the ecosystem [10]. Numerous studies have reported the global dissemination of microplastics and their detrimental effects on the environment and human health. The dreadful effects of MP have been associated with its distinctive features, such as shapes, colors, and varied chemical composition that includes additives and adsorbents [11]. MP is easily readily taken up by organisms owing to its small size, which in turn leads to concentration-dependent accumulation in their internal organs. Previous literatures have reported the harmful effects of microplastics on cells and tissues, including mitochondrial dysfunction and DNA damage. A recent study on polystyrene and polyethylene microplastics demonstrated a significant dose-dependent decrease in cell viability of MDCK and L929 cells in vitro at different time intervals [12]. Another study reported the uptake and localization of polymethylmethacrylate beads by HEK293, A549, and MRC5 cells [13]. However, a research gap still exists regarding the potential effects of polymers on cell signaling and their contributing factors. The severe toxicological effects of MP have been observed and addressed in their synergistic combination with emerging contaminants such as nanomaterials [14]. Recent reports on association of MP and emerging contaminants in the environment have deduced the impact of co-exposure and bioaccumulation in aquatic and terrestrial organisms. The aforementioned study has observed antibiotics, perfluoroalkyl compounds and triclosan showing high sorption capacities towards MPs, imparting toxic effects in varying synergistic or antagonistic ways [14]. Another study has investigated the influence of GO on the toxicity of polystyrene MP on the marine microalgae since GO was intensively exploited for environmental remediation purposes to clean up soil and water contamination [15]. Henceforth, it can be hypothesized that accumulated MP at various ecosystem levels from different sources can bind with nanomaterials like Graphene oxide, under the influence of natural environmental conditions like solar radiation, differential pH, and ionic imbalances [16]. Subjected to thorough research and reporting in both in vitro and in vivo models, these formed conjugates or hybrids may lead to a factual impact on human as well as environmental well-being in a synergistic or antagonistic way.
Macrophages play a pivotal role in innate immunity by regulating many homeostatic and evolutionary host-defense immunological responses. Additionally, macrophages also participate in several other biological processes, such as endogenous levels of reactive oxygen species (ROS), maintaining iron homeostasis, repairing tissue damage, and a list of other metabolic functions [17]. Macrophages have three essential roles to play: antigen presentation, phagocytosis, and immunomodulation. Under various pathophysiological conditions, they are crucial for carrying out normal immune responses. The RAW 264.7 cell line serves as a robust in vitro model for murine macrophages, fundamental to the realm of immunological research. Therefore, determining the biocompatibility of GO@MP can provide insight into its physiological behavior for biomedical and clinical approaches.
An experimental and computational study has been developed to mimic the formation of GO and MP hybrid material at the lab scale to unravel the information regarding the combined and comparative toxicity of GO and MP. At the cellular and molecular level, their combined toxicity is assessed, addressing the toxicity impact of GO and MP. The findings were gathered to answer the raised concern of synergistic toxicity of GO and MP using the RAW 264.7 murine macrophage cell line as the in vitro model and provide new perspectives for toxicological research.
Materials and methods
2
Synthesis of Graphene oxide-polystyrene microplastic (GO@MP) hybrid
2.1
Commercial Graphene oxide nanosheets were purchased from Tokyo Chemical Industry Co., Ltd. (TCI), India. After-usage flow cytometry (FACS) microplastic beads from BD Life Sciences, USA; were used for the synthesis. A speculative hybrid of Graphene oxide (GO) - polystyrene microplastic (MP) was prepared in the laboratory setup using a green methodology to mimic natural environmental conditions (Fig. 1). Freshly prepared floral extract of Calotropis gigantea was used as a catalytic agent. Flowers of Calotropis gigantea were collected early in the day from the premises of the KIIT University campus to procure floral extract. Flowers were washed and weighed (25 g) followed by fine mincing. Further, it was boiled in 500 mL of distilled water for 30-40 min till the appearance of pink color. The obtained solution was then cooled down at room temperature and filtered through a muslin cloth for extraction. With an overnight incubation at 37^0^ C, the synthesis reaction was set up by mixing the GO and MP solution in a ratio of 1:1 (V/V). Followed by incubation, the reaction setup was maintained under UV exposure in a UV crosslinker for two intervals of 30 min each at 100 rpm rotating condition [18]. Consequently, the solution mixture was subjected to washing twice to eliminate unutilized biomolecules in the solution.Fig. 1. Schematic diagram displaying the green synthesis of Graphene oxide- Microplastic (GO@MP) hybrid. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 1
Physiochemical characterization of GO, MP, GO@MP hybrid
2.2
GO, MP, and GO@MP hybrid was characterized by standard physical techniques for their physicochemical properties. The visualization and size determination studies were performed by scanning electron microscope (SEM) (Carl Zeiss, Germany) equipped with EDS (Ametek, Germany). To perform the SEM analysis, samples were first dried and placed on silicon wafers, followed by Pd/Au coating using Sputter Counter. Fourier transform infrared (FTIR) spectroscopy measurements were done to analyze the varying chemical composition of GO, MP, and GO@MP. The FTIR spectrum was captured using a Perkin Elmer FT-IR spectrometer RX-I device with an ATR attachment. The spectrum was recorded in the range of 500 to 4000 cm^-^1 at a resolution of 4 cm^−1^. Dynamic Light Scattering (DLS) method was followed for the estimation of hydrodynamic diameter and zeta potential using the Zetasizer instrument (Malvern, UK) in the Holtfreter (HF) medium.
Cell culture and maintenance
2.3
The Macrophage (RAW264.7) adherent cell line was obtained from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in a complete medium having Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (Himedia), 1% L-glutamine, and 1% antibiotics (a combination of 100 μg/ml streptomycin and 100 IU/ml). Maintenance of cells was done under standard culture conditions at 37 °C with 5% CO2. All reagents were purchased from Himedia.
Cell morphology analysis
2.4
Cell morphology analysis was done through microscopic assessment. To perform the assessment, the cultured RAW 264.7 cells were seeded onto the specimens at a density of 1∗10**^4^** cells per well in 24-well plates. The seeded cells were incubated overnight at 37 °C with 5% CO2 for adherence and growth. Further, the cells were exposed to different concentrations (25 μg/ml - 250 μg/ml) of GO, MP, and GO@MP followed by observations at 24 h, 48 h, and 72 h, respectively. The morphology was obtained using a bright field microscope (Evos M5000, Thermoscientific) and images were collected at an interval of 24 h.
MTT assay
2.5
To analyze the cell viability of RAW 264.7 cells in the presence of GO, MP, and GO@MP, respectively, MTT assay was performed. In 200 μl of complete media, cells were grown at a count of 2.5∗10**^4^** cells per well under standard cell culture conditions. Followed by incubation for 24 h the cells were checked for adherence and the media was changed with 200 μl cell medium containing different concentrations of GO, MP, and GO@MP (25 μg/ml - 250 μg/ml). The treated cells were then incubated for 24 h, 48 h, and 72 h respectively, in humidified atmosphere conditions with 5% CO2 at 37 °C. After every interval of 24 h, the incubated cells were washed twice with PBS (pH 7.4) followed by the addition of 10 μl MTT solution (in 10% DMSO). The cells with MTT solution were incubated for 4 h allowing the MTT to be converted to formazan crystals. Furthermore, the formazan crystals were dissolved in a dissolving solution (buffer composition: 11 gm SDS in 50 ml of 0.2 M HCl and 50 ml isopropanol) by placing the plate in gentle shaking condition at 37 °C. To allow complete solubilization the plate was additionally incubated for 15-30 min, and absorbance was measured precisely at 570 nm using an ELISA plate reader (Epoch, Biotek, Germany). While determining 100% cellular viability, the mean absorbance of untreated cells was used as a reference point and the amount of color product formed was taken to be proportional to the number of viable cells. The experiment was conducted thrice in triplicates to ensure statistical analysis.
Uptake analysis
2.6
To estimate and evaluate the accumulation and internalization of GO, MP, and GO@MP by RAW 264.7 cells, Flow cytometry technique was utilized [19]. RAW 264.7 cells were seeded in 24 well cell culture plates in presence of complete medium at a cell density of 50∗10**^4^** cells/well. After incubation for 24 h, 48h, and 72h, cells were treated with GO, MP, and GO@MP in a similar manner as explained previously in the morphological analysis section. Following that, trypsinization was performed, and cells were washed twice with 1X PBS. The Attune acoustic focusing cytometer (Applied Biosystems, Life Technologies) with a 488 nm argon laser was used to measure the mean side scatter. The SSC analysis was conducted using a logarithmic configuration. FCS Express 7 was used to process data (DeNovo, Los Angeles, CA).
Mitochondrial activity analysis
2.7
The mitochondrial activity of GO, MP, and GO@MP exposed RAW 264.7 cells were evaluated using fluorescent microscopy. MitoTracker™ (Thermofisher Scientific, USA) was used to stain the exposed cells to estimate the mitochondrial activity. In brief, 24h, 48h, and 72h exposed cells were stained with MitoTracker^TM^ (Thermo scientific) dye to stain active mitochondria inside cells. The staining was performed using 2 μM MitoTracker^TM^ in 24-well plate of media volume 500 μL. the extra stains were washed after staining. The images were captured using EVOS fluorescent inverted microscope (ThermoScientific, USA) and processed using Image J. software.
Steatosis analysis
2.8
In order to evaluate the neutral lipid metabolism in RAW 264.7 cells, the standard fluorescent microscopy method was followed to process the treated cells. Precisely, the cells were fixed with 2% paraformaldehyde and permeabilization was done with 0.1% triton X (Himedia, India). Further, for microscopic analysis, cells were stained with BODIPY^TM^ (Invitrogen) dye which is used to stain neutral lipid present inside cells. The images were captured using EVOS fluorescent inverted microscope (ThermoScientific, USA) and processed using Image J. software.
Apoptosis analysis using AO/EtBr staining
2.9
To study the cell death in RAW 264.7 cells treated with GO, MP, and GO@MP apoptosis analysis was carried out using Acridine orange (AO)/Ethidium bromide (EtBr) labeling. Standard techniques like fluorescence microscopy and flow cytometry were employed for apoptosis study. For fluorescence microscopy analysis, cell seeding and treatment were done in a similar way as previously described in the morphological analysis section. Following treatment, the cells were observed and recorded at every 24 h interval till 72 h. Subsequently, the medium containing treatment was removed, and cells were washed thrice with 1X PBS. To process the cells for imaging, the staining was done with Acridine orange (AO) and Ethidium Bromide (EtBr). The images were captured using EVOS fluorescent inverted microscope (ThermoScientific, USA) and processed using Image J. software.
Apoptosis was also estimated using flow cytometry, where RAW 264.7 cells were seeded at a cell density of 50∗10**^4^** cells per well in a 24-well cell culture plate. Incubation and treatment with GO, MP, and GO@MP were done in a similar way as previously described in morphological analysis section. Apoptosis was analyzed at every interval of 24 h to 72 h. The incubated cells were trypsinized and washed with 1X PBS. The Attune acoustic focusing cytometer (Applied Biosystem, Life Technologies) equipped with 488 nm argon laser was used for data collection at 10000 cell count. The processing of data was done in FCS Express 7 (DeNovo, Los Angeles, CA).
Annexin V-FITC/PI assay
2.10
Apoptosis was quantified using an Annexin V–FITC/propidium iodide (PI) kit (Imagenx, Bhubaneswar, India) according to the manufacturer's instructions. RAW 264.7 cells were seeded in 24-well plates at 1.0 × 10^4^ cells/well in complete DMEM and allowed to adhere for 24 h at 37 °C, 5% CO_2_. Cells were then exposed for 24 h to GO, MP, or GO@MP at the indicated concentrations (50 μg/mL and 250 μg/mL) in 500 μL DMEM per well. After exposure, cultures were rinsed three times with 1 × PBS, stained with Annexin V–FITC and PI per kit protocol, and immediately analyzed on an Attune acoustic focusing cytometer (Applied Biosystems/Life Technologies) equipped with a 488 nm laser. 10,000 events were acquired per sample, and data were processed in FCS Express 7 (DeNovo, Los Angeles, CA). Experiments were performed in three independent biological replicates.
Caspase activity analysis
2.11
Caspase-3 activity was quantified using the Merck Caspase-3 Colorimetric Activity Assay Kit (APT165) following the manufacturer's instructions. Briefly, untreated and exposed RAW 264.7 cells were harvested and lysed on ice; clarified lysates (centrifugation 10–15 min, 4 °C) were collected and protein content determined (BCA). Equal protein (typically 50–100 μg per reaction) was incubated in assay buffer with the Ac-DEVD-pNA substrate at 37 °C (60–120 min) in 96-well plates. Hydrolysis of the substrate liberates p-nitroaniline (pNA), which was measured at 405 nm (ε_mM = 10.5). pNA concentration was calculated from the kit's standard curve and reported as pmol pNA·min^−1^·mg^−1^ protein (or as normalized A405 where stated). Each condition was run in triplicate and repeated in three independent experiments.
In silico analysis
2.12
The molecular interaction between graphene oxide (GO) nanosheets and styrene was analyzed using an in silico approach. Molecular docking studies were conducted using AutoDock Vina (Version 1.5.7) [20], where the GO nanosheet was used as the receptor and styrene as the ligand. Prior to docking, both the receptor and ligand structures were optimized using the AutoDock module of MGLTools, Avogardo and ACD/Chemsketch. The grid box dimensions were set to 126 × 126 × 126 and spacing with 0.375 Å. Docking results were analyzed to identify the optimal binding conformation with the lowest binding energy. Post-docking analysis and visualization of molecular interactions were performed using Discovery Studio Visualizer and ChimeraX [21]. Additionally, LigPlot+ was employed to generate 2D interaction plots, providing insights into the molecular interactions and conformational changes in the GO nanosheet upon binding with styrene [22]. The molecular interaction of proteins BCL2, Caspase3, peroxisomal membrane proteins PEX5 and PEX14 with graphene oxide (GO) nanosheets and styrene microplastic was also conducted through molecular docking approach using AutoDock Vina (Version 1.5.7), with BCL2, Caspase3, PEX5, and PEX14 as the receptors and GO nanosheet and styrene as ligands. Uniprot (ID's – P10417, P70677, O09012, Q9R0A0) was utilized to obtain the 3D structures for BCL2, Caspase3, PEX5 and PEX14. The grid box dimensions were set at 100 × 100 × 100, with a spacing of 1 Å. The optimal binding conformation with the lowest binding energy was obtained. Discovery Studio Visualizer and ChimeraX were used for the post-docking analysis and visualization of molecular interactions. 2D interaction plots were generated using LigPlot + to interpret the molecular interactions and conformational changes.
Statistical analysis
2.13
GraphPad Prism v9.5.1 (San Diego, CA) was used for statistical analysis. All the experiments were conducted thrice and in triplicate. Two-way ANOVA was used to evaluate the significance of the differences between the groups. The differences between individual groups were evaluated using Turkey's post hoc analysis.
Results and discussion
3
Green synthesis of GO@MP hybrid
3.1
The synthesis of GO@MP was performed by a green synthesis approach through a lab-mimic process. A freshly prepared floral extract of C. gigantea was used in the synthesis procedure as shown in Fig. 1. After GO and MP were incubated along with the floral extract, a slight greenish hue was observed. On further exposure to UV light, a black color suspension was obtained, followed by the formation of black precipitate, indicating the creation of GO@MP hybrid. The initial mixing of GO and MP with a reddish-pink floral extract of C. gigentea could be the possible reason behind the appearance of a greenish hue. Our earlier research has demonstrated the presence of active biomolecules in the floral extract of C. gigentea determined through Gas-chromatography (GC-MS) analysis [23]. From the GC-MS report, the biomolecules present in the floral extract of C. gigentea include Folic acid, 5-hydroxymethyl furfural bearing -OH group, and saccharides like 6-acetyl b-D-mannose (Table S1). The development of the black precipitate signifying the formation of GO@MP hybrid can be attributed to the reaction facilitated by the help of UV radiation and the biomolecules present in the floral extract of C. gigentea [18].
Physicochemical characterization of GO@MP
3.2
The physicochemical characterization of GO, MP, and GO@MP was done by standard techniques, like spectroscopy and microscopy. As shown in Fig. 2A, the visual inspection trough a stereomicroscope revealed GO@MP as a hybrid structure of GO and MP; the structure was comparable to the sheet-like structure of GO and spherical MP. Further, SEM analysis was performed for detailed visualization. A clear demarcation between the sheet structure of GO and the spherical morphology of MP was obtained with an embedded MP structure with GO sheet in the case of GO@MP (Fig. 2B). The hybridization of MP with the GO sheet was observed as clear evidence of the formation of the GO@MP hybrid. Energy Dispersive X-ray Spectroscopy (EDS) analysis confirmed the presence of C, O, and Si, indicating the presence of GO and MP. However, the silicon wafer substrate can be reasoned to be responsible for the trace amount of silicon discovered in the experimental setup (Fig. S1). The size and stability were further estimated by the determination of the hydrodynamic diameter and zeta potential, using dynamic light scattering techniques. As shown in Fig. 2C, the hydrodynamic diameter of GO, MP, and GO@MP were obtained as 1117.0 ± 374.0 nm, 222.9 ± 22.6 nm, and 1433.0 ± 268.0 nm. Although the measured hydrodynamic diameter of the GO@MP hybrid (∼1433 ± 268 nm) appears larger than the conventional nanoscale range, cellular penetration at such dimensions is still feasible through multiple mechanisms. Macrophages, in particular, are professional phagocytes capable of engulfing particles in the sub-micron to several-micron range via phagocytosis, micropinocytosis, or receptor-mediated endocytosis. Studies have reported polystyrene with diameters of 50-500 nm can rapidly internalize into macrophages and exhibit acute toxicity in cellular activity [24]. Researchers have investigated the interactions between THP-1 macrophages and GO of different sizes (GO of size 500–5000 nm [25]. Therefore, despite their comparatively large hydrodynamic diameter, the GO@MP hybrids are phagocytosed by RAW 264.7 cells, as evidenced by the flow-cytometric side-scatter increase and intracellular localization signals observed in this study. This indicates that cellular entry of GO@MP occurs predominantly through active endocytic pathways rather than passive diffusion. The hybridization of GO with MP explains the larger diameter of GO@MP in comparison to GO as observed [18]. Further, the stability of the synthesized hybrid was studied by determining its zeta potential. As shown in Fig. 2D, the zeta potential of GO, MP and GO@MP was found to be as −68.0 ± 16.8 mV, −41.8 ± 7.0 mV and −47.3 ± 5.7 mV, which demonstrated the stability of the materials and the hybrid material, the hybridized MP with the GO sheet is responsible for the higher value in case of GO@MP as compared to GO. The Fourier transform infrared (FTIR) spectral profiles of MP, GO, and the GO@MP hybrid were presented in Fig. 2E. In the MP spectrum, the characteristic vibrational bands at 1015 cm^−1^ correspond to the C–OH stretching frequency of alcoholic or phenolic groups was found, while the peak at 1655 cm^−1^ can be attributed to C=C stretching vibrations from the aromatic rings. Peaks at 2846 and 2948 cm^−1^ result from symmetric and asymmetric aliphatic C–H/CH_2_ stretching vibrations, indicating hydrocarbon chains typical of polymeric materials. A broad band at 3396 cm^−1^ relates to O–H stretching, likely arising from carbohydrates, proteins, and polyphenols on the MP surface. The GO spectrum displays a distinct peak at 1032 cm^−1^ for C–O stretching, while the peaks at 1571 cm^−1^ and 1712 cm^−1^ represent C=C bonds and C=O stretching vibrations due to carboxyl groups, respectively [26]. The broad peak observed around 3096 cm^−1^ can be associated with aromatic or vinylic C–H stretching. The GO@MP hybrid showed a band at 1031 cm^−1^ for C–O stretching, indicating a negligible shift from GO due to the retention of epoxide or hydroxyl groups. However, the appearance of peaks at 1396 cm^−1^ for C–O bending frequency and a shifted C=C peak at 1588 cm^−1^ suggested possible π–π interactions between the GO sheets and MP components. These spectral shifts and merged features confirm the successful integration of GO onto MP through non-covalent interactions, resulting in a GO@MP composite structure [18,27]. Further, the molecular docking analysis was done to understand the molecular interaction and confirmation of GO and MP forming GO@MP. Fig. 3 shows the molecular interaction of GO and MP in GO@MP conjugate. The molecular docking study revealed styrene binds to the GO nanosheet with a binding affinity of −9.0 kcal/mol. The interaction occurs through multiple atomic contacts, primarily involving π-alkyl and π-cation interactions. The observed interaction distances were 5.07 Å (Pi-Alkyl), and other interaction types are Pi-cation with distances 4.32 Å, 3.86 Å, 3.71 Å, 4.19 Å, and 4.59 Å. 2D Visualization using LigPlot + revealed structural changes in the GO nanosheet upon interaction with styrene, indicating potential modifications in its electronic and conformational properties.Fig. 2. Physicochemical characterzation of the GO, PS and GO@MP; (A) Optical microscopy image of the (a) Graphene oxide (GO) (b) Polystyrene Microplastic (MP) (c) GO@MP; (B) Scanning electron microscopy images of (a) GO (b) MP and (c) GO@MP; (C) hydrodynamic diameter of GO, MP and GO@MP determined by dynamic light scattering; (D) Zeta potential of GO, MP and GO@MP determined by dynamic light scattering; (E) FTIR spectrum of GO, MP and [email protected]. 2. Fig. 3Molecular interaction of GO and MP in GO@MP hybrid as determined by computational molecular docking.Fig. 3
In vitro cytotoxicity of GO@MP
3.3
RAW 264.7 murine macrophage cell lines have been widely reported to understand the molecular mechanisms underlying phagocytosis [28]. Hence, the cell lines were chosen to estimate the cytotoxicity of GO@MP and its molecular impact. The morphological changes occurring in RAW 264.7 cells upon exposure to different concentrations (25μg/ml- 250 μg/ml) of GO, MP, and GO@MP were observed at 24h, 48h, and 72h of exposure using a bright field microscope. While the environmental concentrations of microplastics (MP) and Graphene Oxide (GO) in natural waters or human bodily fluids are typically reported in the ng/mL to low μg/mL range, the concentration range of 25−250 μg/mL used in this in vitro macrophage study in context of the two primary objectives: first, to establish a clear dose-response relationship for the materials, and second, to investigate the maximum potential toxicity and underlying cellular mechanisms of the GO@MP hybrid.
As shown in Fig. 4(A)(B)(C), at higher concentrations of exposure of GO, MP, and GO@MP, the cells were seen to be detached, shrunken, and scattered, with membrane fractioning and cytoplasmic shrinkage during their interaction. Interestingly, after 72h of treatment, proliferation of cells was observed at higher concentrations of MP and GO@MP in comparison to the normal morphology of cells. With increasing exposure concentrations of GO, MP, and GO@MP, the attachments were seen on the cell surface, causing more internalization inside cells, leading to changes in the cellular physiology [29]. The attachment was found to be higher in the case of GO@MP compared to MP and GO at each concentration of exposure. The MTT assay revealed a dose-dependent and time-dependent survivability of cells. The results were in alignment with the studies done by Wang et al., which observed a decrease in viability of RAW264.7 cells with increasing concentration of MP ranging from 0.2 to 1.5 mg/ml [30]. As shown in Fig. 4(D), (E), (F), the cell viability of RAW 264.7 was observed to decline with an increase in exposure concentration of GO, MP, and GO@MP. Comparatively, cells were less viable in the case of GO@MP exposure than GO and MP at each exposure concentration. The LC_50_ of GO, MP, and GO@MP was calculated as 429.9 ± 14 μg/mL, 334.0 ± 14 μg/mL, and 193.3 ± 14 μg/mL for 24h, while it was 262.1 ± 14 μg/mL, 172.9 ± 14 μg/mL, and 123.6 ± 14 μg/mL for 48h exposure (Fig. S2). Consequently, the LC_50_ for 72h was calculated as 23.2 ± 14 μg/mL, 21.9 ± 14 μg/mL, and 7.5 ± 14 μg/mL for GO, MP, and GO@MP exposure. The exhibition of morphological abnormalities and cell survivability was hypothesized as a result of the accumulation and internalization of GO, MP, and GO@MP at a differentiated level. Hence, the accumulation and internalization were estimated through the change in granularity of the cells by measuring side scatter intensity using flow cytometry [27]. As shown in Fig. 4(G), (H), (I), the side scatter intensity of GO, MP, and GO@MP was observed as GO < MP < GO@MP, both at 50 μg/ml (Lower) and 100 μg/ml (higher) of exposure concentration. Interestingly, the pattern was similar at 24h, 48h, and 72 h of exposure. Previous studies have reported the interaction and internalization of nanoparticles inside cells [29], attribution to the abnormal cell physiology through endocytosis, and disturbed physiology [31,32]. The comparative accumulation and internalization of GO, MP, and GO@MP referred to their comparable cytotoxicity, with a higher result for GO@MP exposure than GO and MP. The observed effects at the GO@MP concentrations tested do not represent current population-wide environmental risks. Instead, these data serve as a mechanistic alert demonstrating the intrinsic toxic potential of the hybrid structure. The pronounced effects at 250 μg/mL confirm that high-dose, local exposure, such as through occupational inhalation or pathological tissue accumulation, poses a significant hazard and warrants further in vivo and chronic low-dose studies.Fig. 4In vitro toxicity of GO, MP, and GP@MP; Morphological changes in RAW 264.7 cells exposed to GO, MP and GO@MP for (A)24h (B) 48h (C)72h; Cell viability of RAW264.7 cells exposed to GO, MP and GO@MP for (D)24h (E) 48h (F)72h determined by MTT assay, the values represent the mean ± SD of three independent experiments. ∗P > 0.5, ∗∗P > 0.01, and ∗∗∗∗P > 0.001 as obtained from post hoc analysis after one-way ANOVA; Side scatter analysis of RAW264.7 cells exposed to GO, MP and GO@MP for (G)24h (H) 48h (I)72h determined by Flow cytometry determining the accumulation and internalization.Fig. 4
Cellular toxicity
3.4
The attachment and internalization of GO, MP, and GO@MP were hypothesized to influence the cellular physiology of the macrophage cells (RAW 264.7) [18,19]. Mitochondrial activity is one of the key components of cellular physiology owing to its role in ATP generation and induction of reactive oxygen species. GO, MP, and GP@MP were hypothesized to influence the induction of oxidative stress through abnormal mitochondrial activity. Previous literatures suggest GO nanosheets induce mitochondrial fragmentation by rupturing cell membrane thereby disrupting mitochondrial homeostasis leading to autophagy and apoptosis in different mammalian cells [[33], [34], [35]]. Similarly, polystyrene MPs have been associated with mitochondrial toxicity in mice [36] as they damage mitochondrial structure via oxidative stress and upregulate both intrinsic and extrinsic apoptotic pathways [37]. The hypothesis was checked by measuring the fluorescent intensity of Mitotracker ^TM^CM Rox stained in the GO, MP, and GO@MP exposed cells. As shown in Fig. 5A, (B), (C), the red fluorescence of the dye was found to increase with an increase in exposure concentration of GO, MP, and GO@MP, indicating a concentration-dependent effect on mitochondrial activity, induced oxidative stress in cells. Interestingly, the quantitative assessment showed higher mean fluorescent intensity in the case of GO@MP exposure compared to GO and MP at each concentration and exposure time. In case of GO exposure, the mean fluorescent intensity was significantly higher than the MP exposure, advocating the fact that the oxidative stress induction due to mitochondrial activity was influenced in a pattern of GO < MP < GO@MP [38]. The change in concentration-dependent mitochondrial activity can be attributed to the differential accumulation and internalization of GO, MP, and GO@MP [39]. The differential oxidative stress can be attributed to the induction of hypoxic conditions due to clogging of the cell membrane and the interaction of GO, MP, and GO@MP with the membrane and physiological proteins. Moreover, the higher induction of oxidative stress and mitochondrial activity in the case of GO@MP can be attributed to the synergistic effect of GO and MP [18].Fig. 5In vitro impact of GO, MP, and GO@MP on RAW264.7 cells. Fluorescent image of Mito Tracker Red CMXRos^TM^ stained cells showing comparative analysis of induced mitochondrial activity in cells exposed to GO, MP, and GO@MP for (A) 24h, (B) 48h, (C) 72h. Comparative Mean fluorescent intensity of MitoTracker Red CMXRos^TM^ in RAW264.7 cells exposed to GO, MP, and GO@MP for (D) 24h, (E) 48h, (F) 72h. The values represent the mean ± SD of three independent experiments. ∗P > 0.5, ∗∗P > 0.01, and ∗∗∗∗P > 0.001 as obtained from post hoc analysis after one-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 5
Further, to estimate the mechanistic and differential cellular toxicity of GO, MP, and GO@MP, it was speculated that their cellular interaction may lead to influential regulation on other physiological processes like lipid metabolism and apoptosis. Previous reports have suggested the effect of GO and MP on lipid metabolism of cells. Moreover, it has been reported that the excessive lipid peroxidation alters the certain membrane protein characteristics and further modifies the cellular physiology [39,40]. With reference to these facts, it was speculated that the differential interaction of GO, MP, and GO@MP may lead to discrepancies in lipid metabolism and generation of low-density (LDL) and very low-density lipids (VLDL). The speculation was crisscrossed experimentally by steatosis analysis through BODIPY stain in macrophages exposed to GO, MP, and GO@MP at different concentrations. BODIPY has been recognized to stain neutral lipid droplets like LDL and VLDL in cells exhibiting green fluorescence. As shown in Fig. 6A,(B),(C), cells treated with GO, MP, and GO@MP for 24h, 48h, and 72h exhibited concentration-dependent and time-dependent green fluorescence. Comparatively, the fluorescence intensity was found to be highest in the case of GO@MP exposure compared to GO and MP, followed by MP and GO exposures (Fig. 6(D),(E),(F)). The trend was followed at 24h, 48h, and 72h exposure. The results indicated the induction of steatosis in macrophages on exposure to GO, MP, and GO@MP, and can be ascribed to the influential effect on the structure and function of lipid metabolism proteins like PEX protein due to intrinsic molecular interactions. Moreover, the highest effect shown by GO@MP can be attributed to the cooperative effect of GO and MP, leading to hyperactivity in the transport of LDL and VLDL to the circulatory system for subsequent tissue transfer [41].Fig. 6In vitro impact of GO, MP, and GO@MP on RAW264.7 cells. Fluorescent image of BODIPY-stained cells showing comparative analysis of LDL and VLDL in cells exposed to GO, MP, and GO@MP for (A) 24h, (B) 48h, (C) 72h. Comparative Mean fluorescent intensity of BODYPY in RAW264.7 cells exposed to GO, MP, and GO@MP for (D) 24h, (E) 48h, (E) 72h. The values represent the mean ± SD of three independent experiments. ∗P > 0.5, ∗∗P > 0.01, and ∗∗∗∗P > 0.001 obtained from post hoc analysis after one-way ANOVA.Fig. 6
The phenomenon of steatosis and oxidative stress has been defined to play an important role in the process of cell death, processes like apoptosis [42]. Additionally, researchers has suggested that GO induces autophagy and apoptosis via ROS-dependent pathway in colorectal cancer cells [43]. Owing to the data obtained for the induction of steatosis and oxidative stress, by GO, MP, and GO@MP at differential levels, the results indicated the differential action of GO, MP, and GO@MP on nucleic acid damage and apoptosis of cells. The hypothesis was investigated through Acridine Orange-Ethidium Bromide (AO-EtBr) staining using fluorescence microscopy on the GO, MP, and GO@MP exposed macrophage cells for 24h, 48h and 72h. AO/EtBr staining effectively differentiates viable (green fluorescence) from apoptotic or necrotic (red fluorescence) cells, providing a clear visual and quantitative measure of membrane integrity and cell viability [44]. As shown in Fig. 7A, (B), (C), the results showed a clear concentration-dependent increase in cytotoxicity across all treatment groups, with GO@MP exhibiting the most severe effects. At lower concentrations (25–50 μg/mL), cells treated with GO or MP alone showed moderate changes, including a mix of viable and early apoptotic cells. However, cells exposed to GO@MP at the same concentrations displayed a marked increase in yellow to red fluorescence, indicative of early to late apoptosis and necrosis. This trend was exacerbated at higher concentrations (100–250 μg/mL), where GO@MP-treated cells exhibited extensive red fluorescence, reflecting severe membrane damage and widespread cell death. These findings were corroborated by quantitative analysis of fluorescence intensity (Fig. 7(D), (E), (F)), which demonstrated a statistically significant increase in red and a decrease in green signal in GO@MP-treated groups compared to GO or MP alone. The fluorescence imaging was verified by flow cytometry analysis [45]. As shown in Fig. 8, the flow cytometry data represented the apoptotic/necrotic cell populations after treatment with GO, MP, and GO@MP at 50 μg/mL for 24, 48, and 72 h, using acridine orange (AO) and ethidium bromide (EtBr) staining. Viable cells appear in the lower left quadrant (AO^−^/EtBr^−^), early apoptotic cells in the lower right (AO^+^/EtBr^−^), late apoptotic or necrotic cells in the upper right (AO^+^/EtBr^+^), and necrotic cells in the upper left (AO^−^/EtBr^+^). Untreated control cells show >98% viability at all time points, indicating baseline health. GO and MP treatment alone cause a moderate increase in early apoptotic cells over time, with GO showing 14.84% and MP 15.54% early apoptosis at 72 h. However, GO@MP treatment results in the highest levels of early and late apoptotic cells, 33.02% early apoptosis at 24 h, followed by sustained apoptosis (13.94%) and increased late apoptosis/necrosis (17.93%) at 72 h. Similarly, the exposure at 250 μg/mL concentration revealed significant cytotoxic effects of GO, MP, and particularly the GO@MP composite over time (Fig. 9). Untreated cells showed >98% viability across all time points, indicating assay consistency. GO exposure caused a marked shift from viable cells (92.17% at 24 h) to early apoptotic (36.20% at 48 h) and late apoptotic/necrotic populations (1.22% at 72 h), suggesting progressive membrane damage and apoptosis. MP treatment induced a more immediate cytotoxic response, with early apoptotic cells rising from 52.84% at 24 h to 29.75% at 48 h, followed by increased necrotic cells (1.89%) at 72 h, indicating sustained cellular stress. The GO@MP composite exhibited the most severe effects. At 24 h, early apoptosis was already elevated (11.20%), progressing sharply to 42.74% at 48 h. By 72 h, early and late apoptotic/necrotic populations reached 22.53% and 2.62%, respectively, while viable cells dropped to 74.63%.Fig. 7In vitro impact of GO, MP, and GO@MP on RAW264.7 cells. Fluorescent image of Acridine Orange/EtBr stained cells showing comparative analysis of apoptosis in cells exposed to GO, MP, and GO@MP for (A) 24h, (B) 48h, (C) 72h. Comparative Mean fluorescent intensity of Acridine Orange in RAW264.7 cells exposed to GO, MP, and GO@MP for (D) 24h, (E) 48h, (E) 72h. The values represent the mean ± SD of three independent experiments. ∗P > 0.5, ∗∗P > 0.01, and ∗∗∗∗P > 0.001 obtained from post hoc analysis after one-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 7. Fig. 8In vitro impact of GO, MP, and GO@MP on RAW264.7 cells. Flow cytometry analysis of Acridine Orange/EtBr-stained cells showing comparative analysis of apoptosis in cells exposed to 50 μg/mL of GO, MP, and GO@MP for (A) 24h, (B) 48h, (C) 72h. The analysis was performed by using FCSexpress 7. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 8. Fig. 9In vitro impact of GO, MP, and GO@MP on RAW264.7 cells. Flow cytometry analysis of Acridine Orange/EtBr-stained cells showing comparative analysis of apoptosis in cells exposed to 250 μg/mL of GO, MP, and GO@MP for (A) 24h, (B) 48h, (C) 72h. The analysis was performed by using FCSexpress 7. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 9
These results indicate that GO@MP induces significantly higher apoptotic responses compared to GO or MP alone, particularly at earlier time points, suggesting a rapid onset of cytotoxicity. The data also suggest sustained and cumulative toxicity over 72 h, reinforcing that the GO@MP hybrid poses greater cellular stress and damage than GO and MP. Further, the results were verified using the Annexin V/FITC assay. Annexin V–FITC/PI cytometry identifies apoptosis as the dominant response at moderate exposures, with a necrotic shift at the most injurious conditions. As shown in Fig. S3, the dot plots showed >99% viable cells in untreated controls (lower-left quadrant, LL: 99.9%; early apoptosis, lower-right quadrant, LR: 0.0%; late apoptosis/secondary necrosis, Upper right, UR: 0.0–0.1%; primary necrosis, Upper left, UL: 0.0–0.1%). Upon exposure, two consistent patterns emerged. First, at a lower concentration of 50 μg/mL, the early-apoptotic (Annexin V^+^/PI^−^; LR) and late-apoptotic (Annexin V^+^/PI^+^; UR) fractions increased while primary necrosis (Annexin V^−^/PI^+^; UL) remained low. It was observed in LR ≈ 6.5–8.4% with UR ≈ 2.4–4.4% and LL falling to ≈88.7–93.9%. Second, under the high concentration exposure (250 μg/mL), the profile shifted toward PI-only populations (UL ≈ 11.4–16.7%) with minimal LR/UR and LL ≈ 83–88%, consistent with rapid membrane rupture/primary necrosis.
The results were aligned with AO/EtBr fluorescent imaging and flow cytometry assay, and indicated to the higher occurrence of necrosis and apoptosis in the case of GO@MP exposure compared to GO, MP exposure. Further, the justification was carried out by using the caspase activity analysis of the GO, MP, and GO@MP exposed for 24h. As shown in Fig. S4, Colorimetric caspase activity (405 nm) increased monotonically with concentration for all materials (0 → 25 → 50 → 100 → 250 μg/mL). At each matched dose, GO@MP showed the highest activity, MP was intermediate, and GO was lowest. Group differences were modest at 25 μg/mL (GO vs MP, n.s.; GO@MP vs GO/MP, small but significant), but became highly significant from 50 μg mL^−1^ onward (two-way ANOVA with Tukey, ∗∗∗∗P < 0.0001 for GO@MP vs GO; frequently P < 0.001–0.0001 for GO@MP vs MP). At 100 and 250 μg/mL the GO@MP response clearly diverged, consistent with amplified activation of executioner caspases relative to single-component exposures.
The biochemical results independently validated the Annexin V–FITC/PI shifts, MTT viability loss, mitochondrial dysfunction (MitoTracker), uptake/SSC increase, and steatosis (BODIPY), supporting mitochondria-linked apoptotic death as the dominant pathway. The heightened toxicity of GO@MP may be attributed to a synergistic interaction between the GO and MP particles. GO's sharp edges and oxidative properties, combined with MP's persistence and possible leachates, may jointly exacerbate cellular stress responses, leading to elevated reactive oxygen species (ROS), mitochondrial dysfunction, and compromised membrane integrity. This aligns with the experimental results and previous reports that co-exposure scenarios can induce greater biological disruption than single-agent exposures.
In silico analysis
3.5
The experimental results underscored the combined and synergistic cytotoxic effect of GO and MP, indicating the need for integrated mechanistic assessments of GO@MP hybrid effects. Concerning the facts, the mechanistic cytotoxicity was speculated to be deduced at the molecular level due to the intrinsic molecular interaction of GO, MP, and GO@MP with metabolic proteins, playing an important role in oxidative stress, steatosis, and apoptosis [18]. Molecular docking simulations were employed to investigate the interactions between GO and polystyrene (MP) and the peroxisomal import receptor proteins PEX5 and PEX14, which play pivotal roles in the translocation of matrix proteins into peroxisomes, leading to lipid metabolism [46]. As shown in Fig. 10, the results revealed distinct binding behaviors depending on the type of nanomaterial and protein involved. Graphene exhibited strong and spatially extensive interactions with both PEX14 and PEX5. In the case of PEX5 (Fig. 10A), graphene formed multiple stabilizing π-π stacking interactions, particularly involving key residues such as Trp159 and Gln52, with a binding affinity of −13.1 kcal/mol, suggesting a potential to interfere with protein-protein recognition domains essential for peroxisomal docking (Figs. S5 and S6, Table 1). The corresponding 2D interaction map confirmed broad contact with the protein surface, likely to result in conformational modulation or inhibition of function. Conversely, polystyrene exhibited comparatively weaker and more localized binding to PEX5 (Fig. 10B, S7, Table 1), primarily via hydrophobic interactions involving residues like Asp97 and Leu94, with a binding affinity of −5.4 kcal/mol, indicating a lower potential to disrupt native protein architecture. Similarly, the GO showed strong affinity for PEX14 (Fig. 10C, S8, S9, Table 1), engaging in hydrogen bonding and extensive van der Waals contacts with residues such as Gln420 and Thr117 with a binding affinity of −10.0 kcal/mol. The interaction network suggests a high likelihood of interference with PEX5's cargo recognition or interaction with PEX14 [46]. On the other hand, polystyrene binding to PEX14 (Fig. 10D, S8, S9, Table 1) was limited to surface-level interactions with binding affinity of −4.2 kcal/mol, engaging residues such as Ser1 and Lys453 via weaker π-π interactions. The 2D interaction diagrams corroborate these findings, highlighting the more robust and widespread binding pattern of GO compared to polystyrene (MP). These results suggested that GO, due to its planar aromatic structure and high surface reactivity, may pose a greater risk for peroxisomal dysfunction by interfering with PEX5–PEX14–mediated import mechanisms [47].Fig. 10In silico analysis of GO, and MP with metabolic proteins of cells. Interaction of PEX 5 protein with (A) GO and (B) MP determined by molecular docking.; PEX14 interaction with (C) GO and (D) MP as determined by molecular docking. Post-docking visualization was performed by Discovery studio. 2D analysis of interactive diagram was obtained using LigPlot^+^.Fig. 10. Table 1Details of ligand-protein interaction explaining molecular interaction of different proteins with GO and MP (Styrene).Table 1. Sl. NO.LigandProteinBinding affinityResidues Participating in hydrophobic interactionResidues forming hydrogen bondsResiduesBond lengths (Å)1.GOBCL2−10.9Pro88, Pro87, Thr7, Val89, Pro201, Leu92Tyr92.90Asp1932.562.GOCaspase3−12.5Trp214, Trp206, Phe256, Leu252, Phe250Glu2482.70Ser2513.22 and 3.183.GOPEX5−13.1Tyr163, Trp159, Phe99Ser6122.81Gln1663.074.GOPEX14−10.0Pro17, Pro16, Arg25, Thr59, Val22Pro173.16Glu203.02 and 3.34Gly182.69 and 2.885.StyreneBCL2−4.5Ser102, Val156, Lys22, Gly152, Val153NANA6.StyreneCaspase3−4.8Ser205, Trp206, Phe256, Tyr204NANA7.StyrenePEX5−5.4Pro454, Leu459, Phe415, Leu411NANA8.StyrenePEX14−4.2Leu53, Lys56, Phe52, Phe35, Ala32, Thr31NANA
The experimental analysis of cell death estimation showed a distinctive and collaborative effect of GO and MP, leading to the fact of the dysregulation in functionality of apoptosis-related proteins due to the intrinsic interaction with GO and MP [18]. To uncover the mechanism, an interaction study was performed for proteins BCL2 and Caspase3 with GO and MP. BCL2, an anti-apoptotic protein, and Caspase-3, a critical executioner caspase, are central to the intrinsic pathway of apoptosis [48]. The docking analyses revealed material-specific binding preferences and affinities, with graphene exhibiting markedly stronger and more extensive interactions than polystyrene for both targets. As shown in Fig. 11, GO binds robustly to BCL2 within a hydrophobic groove comprising residues such as Val145, Phe101, and Arg107 with a binding affinity of −10.9 kcal/mol. These interactions are primarily mediated through π-π stacking, hydrophobic forces, and van der Waals contacts, suggesting that the GO could disrupt protein-protein interactions or prevent ligand access to regulatory domains (Fig. 11A, S10, S11, Table 1). The corresponding 2D interaction map confirms this extensive binding interface, supporting the likelihood of functional interference. In contrast, polystyrene interacts more modestly with BCL2 (Fig. 11B, S10, S12, Table 1), involving residues like Lys22 and Tyr21 with a binding affinity of −4.5 kcal/mol through weaker hydrophobic and π-π interactions. The interaction surface suggested the risk of disrupting BCL2's native function. A similar pattern of interaction was observed with Caspase-3. Graphene displayed a significantly highest binding affinity of −12.5 kcal/mol to the protein (Fig. 11C, S13, S14, Table 1), engaging active or regulatory residues such as Phe256 and Trp206. The planar surface of graphene allowed for large contact areas stabilized by aromatic stacking and hydrogen bonding, which could potentially inhibit enzymatic activity or alter substrate recognition. Polystyrene, however, exhibited weaker interactions with Caspase-3 (Fig. 11D, S13, S15, Table 1), forming π-π interactions mainly with Trp206 and Phe256, as reflected by a simpler and more localized 2D interaction map.Fig. 11In silico analysis of GO, and MP with metabolic proteins of cells. Interaction of BCL2 protein with (A) GO and (B) MP determined by molecular docking.; Caspase3 interaction with (C) GO and (D) MP as determined by molecular docking. Post-docking visualization was performed by Discovery studio. 2D analysis of interactive diagram was obtained using LigPlot^+^.Fig. 11
The findings suggested that GO exerts pronounced biological effects along with polystyrene by perturbing apoptosis signalling through both inhibition of caspase function and modulation of anti-apoptotic pathways.
Mechanism
3.6
The study investigated and illustrated the detailed in vitro mechanistic toxicity of GO, MP, and GO@MP hybrid in macrophage cells. The experimental investigation showed differential accumulation and internalization of GO, MP, and GO@MP, leading to abnormal morphology and physiological metabolic processes in a concentration-dependent manner. The cellular mechanistic investigation revealed the effect of GO, MP, GO@MP on lipid metabolism, mitochondrial activity, and apoptosis in a differential manner, attributed to the synergetic effect of GO and MP. The computational analysis unraveled the intrinsic atomic interaction of GO and MP with metabolic protein like PEX5, PEX14, BCL2, and Caspase 3, contributing to the influence on structural and functional regulation of the proteins, leading to the metabolic abnormalities. Previous reports have mentioned the effect of GO and MP on oxidative stress, lipid metabolism, and apoptosis in different cell lines [49] and in vivo models [18]. With reference to the facts and results obtained, the mechanistic differential toxicity of GO, MP, and GO@MP can be caricatured as a consequence of accumulation and internalization of GO, MP, and GO@MP at variable levels. The variable internalized GO and MP interact with metabolic proteins like PEX5 and PEX14 leading to the steatosis in cells at variable level. Moreover, the accumulation at the surface of cells leads to blockage of the cell membrane, leading to hypoxic condition, which is compensated by the abnormal mitochondrial activity. Additionally, the internalized GO and MP interact with apoptotic proteins like Caspase3 and BCL2 to influence their structural and functional integrity. These abnormal conditions at the cellular and molecular level can be attributed to the cell apoptosis. The variability in the cellular physiology and apoptosis in cells exposed to GO and MP is due to their variability in chemical and physicochemical nature. The higher cellular effect in case of GO@MP exposure is due to the synergistic impact of GO and MP. The mechanism can be visualized through the diagram shown in Fig. 12.Fig. 12. Schematic presentation of the mechanistic cellular toxicity of Graphene oxide-Microplastic hybrid (GO@MP) with RAW264.7 cells.Fig. 12
Conclusion
4
In brief, the study elucidates the in vitro cytotoxicity of Graphene Oxide (GO), Polystyrene microplastic (MP), and the Graphene-Microplastic (GO@MP) hybrid in macrophage cells. The hybrid was created in the laboratory using an eco-friendly method to replicate the natural process. The hybrid was postulated to demonstrate cellular toxicity owing to its heterogeneity in physicochemical properties relative to GO and MP. The experimental analysis of the physicochemical characteristics, including size, hydrodynamic size, and Zeta potential of the GO@MP hybrid, demonstrated considerable heterogeneity compared to GO and MP. The computational analysis demonstrated the inherent atomic interaction between GO and MP, resulting in the formation of the GO@MP hybrid. The comparative in vitro mechanistic cellular toxicity was clarified utilizing macrophage cells. The outcome indicated the increased cellular toxicity of GO@MP in macrophages, assessed through morphological, physiological, and metabolic changes at both cellular and molecular levels. The mechanism of cellular toxicity was determined to involve interference in metabolic processes such as steatosis, mitochondrial function, and apoptosis, resulting from the accumulation and internalization of GO and MP interacting with metabolic proteins at varying levels. The results elucidated the cause of the variability in vitro toxicity of GO, MP, and GO@MP. The study revealed significant information regarding the comparative in vitro cellular toxicity of GO, MP, and GO@MP, indicating the critical implications of their harmful effects for future research and the necessary precautions for the use of graphene oxide and polymers for human health.
CRediT authorship contribution statement
Adrija Sinha: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Arghyadeep Mayur: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Snehasmita Jena: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. Aishee Ghosh: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Mrutyunjay Suar: Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Resources, Software, Validation, Writing – original draft, Writing – review & editing. Suresh K. Verma: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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