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Original Article
ARTICLE IN PRESS
doi:
10.25259/AJC_961_2025

Gold nanoparticles on chitosan functionalized graphene oxide: Catalytic reduction of nitroarenes and anti-bladder carcinoma activity

, , ,
 Department of Urology, Shanxi Province Cancer Hospital, Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences, Cancer Hospital Affiliated to Shanxi Medical University, No. 3, Workers New Street, Taiyuan, China

* Corresponding author: E-mail address: qufa_139942@sohu.com (W. Wang)

Received: , Accepted: ,
© 2026 Arabian Journal of Chemistry - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

This work describes a simple and environmentally friendly process for creating a graphene-chitosan/gold (GO-CS/Au) nanocomposite. This green synthesis method includes the reduction of gold ions onto graphene oxide that had previously been functionalized with chitosan, using pomegranate juice as a bio-reductant and capping agent. The pomegranate juice’s natural polyphenolic components helped to stabilize and reduce gold nanoparticles (AuNPs) on the GO-CS surface. To verify the GO-CS/Au nanocomposite’s successful synthesis and structure, a wide range of analytical methods were used. These techniques included X-ray diffraction (XRD), transmission electron microscopy (TEM), inductively coupled plasma (ICP) analysis, energy-dispersive X-ray spectroscopy (EDX) with elemental mapping, and field-emission scanning electron microscopy (FE-SEM). GO-CS/Au nanocomposite was subsequently assessed in vitro for biological applications. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, its potential as an anti-cancer drug was evaluated against two bladder cancer cell lines, 5637 and transitional cell carcinoma, SUPINSKI (TCCSUP). The nanocomposite reduced cell viability in a dose-dependent manner; it’s computed IC50 values were 458 and 648 µg mL-1, respectively. The antioxidant capacity of the GO-CS/Au nanocomposite was also investigated via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which revealed a notable antioxidant activity with an IC50 value of 98 µg mL-1. Furthermore, chemical catalysis of the material was explored in the reduction of different nitroarenes with sodium borohydride, being monitored over UV-Vis. The material demonstrated excellent reusability, holding onto roughly 85% of its conversion efficiency after seven runs.

Keywords

Bladder cancer
Chitosan
Gold NPs
Nitro reduction
Pomegranate

1. Introduction

Gold nanoparticles (Au NPs) are one of the most effective catalysts in a variety of reactions in recent years, such as selective oxidation of alcohols, carbonyls, and cycloalkanes; reduction of nitroaromatics; C–C coupling reactions; epoxidation of propylene; hydrogenation of C=C/O; etc. [1-4]. The exceptional catalytic activity of Au NPs is linked to their shape, surface state, high surface-to-volume ratios, and obviously, the negative redox potential. The high surface energy of free Au NPs, however, makes them prone to aggregation, which results in a certain deterioration of low recyclability, poor durability, and catalytic activity. Immobilizing Au NPs on an appropriate, non-hazardous and reusable support is demonstrated to be a helpful strategy for keeping Au NPs from aggregating together as well as for preserving and enhancing their catalytic and bio-activity [5-7]. Au NPs have been supported over various materials, like metal oxides, carbonaceous materials, clays, polymers, and metal organic frameworks [8-14]. Because of their characteristics and the interactions at the Au NPs-support interface, the supports are always essential to the prepared Au NPs catalyst. The two-dimensional, large surface area carbon compound known as graphene oxide (GO) is one of them. It has several lipophilic oxygenated functional groups that enable it to disperse nicely in water and has been used as a catalyst in a variety of catalytic processes [15]. Furthermore, because of its enormous surface area and superior mechanical, thermal, and electrical properties, GO has been successfully used to deposit or immobilize a variety of species, including metal NPs, oxides, complexes, etc. These assisted catalysts have demonstrated better catalytic activity in most of the reactions that have been examined thus far [15-19].

Nevertheless, to overcome certain limitations, more research is still required on the immobilization of Au NPs on GO, even though numerous studies have been published on surface-modified GO matrix in order to create viable catalytically dynamic hybrid materials. Some limitations include low loading of Au NPs after being immobilized on GO, frequent leaching of Au NPs because of poor interactions between Au NPs and GO, and the use of toxic reductants like hydrazine and sodium borohydride (NaBH₄) for reducing GO and Au NPs. To create graphene-based hybrid materials, a realistic solution to this problem must be implemented. The use of plant-based phytochemicals or biopolymeric agents in conducting the biogenic reduction of metal ions into NPs or facilitating the binding of NPs over the support has been quite familiar these days. To create a stable shell or capsule over GO, we employed chitosan (CS), an eco-friendly reducing and stabilizing biopolymer produced by deacetylation of chitin. Chitin is found abundantly in the skeletons of shellfish, insects, beaks of cephalopods, etc., and is considered the second most naturally occurring polymer following cellulose [20-22]. Further, Au NPs were in situ generated over this composite, being promoted over the phytomolecules from pomegranate juice. The juice contents, e.g., different biomolecules like polyphenols (ellagitannins, anthocyanins, and flavonoids), lignans, triterpenoids, alkaloids, and several organic and phenolic acids, facilitated the in situ biogenic reduction of Au ions to their NPs without employing toxic reductants to afford the GO-CS/Au NPs.

Today, nanotechnology is important in various scientific areas [23-27]. Recently, nanomaterials have been developed as an exciting way to develop unique and useful materials [28-32]. They are also involved in several areas such as the environmental, catalysis, environmental process, and healthcare [33-37]. One of the most concerning chemical pollutants of natural and drinkable water is nitroarenes. The environment is gravely harmed by the many chemicals, dyes, medications, and the textile and paper industries that release various derivatives of this moiety into water bodies like rivers or lakes [38-42]. Consuming this contaminated water seriously damages the liver, kidney, and brain systems of humans and animals [43-46]. Thus, reducing the negative effects of nitroarenes before disposing of them would be a good way to deal with the issue. To reduce nitroarenes, we chose to use GO-CS/Au NPs catalytically with NaBH₄ as the hydride donor. Several nitro substrates were reduced using this catalyst, providing excellent yields in short reaction times. 

It is now expected that the GO-CS/Au nanocomposite will have important biological properties since it is a biogenic material. In recent days, the importance of bio molecularly designed nanoparticles as novel nanomedicine formulations has gained more attention. They have an enormous surface which facilitates effective and fruitful medication administration, and they can precisely target damaged areas. In addition, they are tiny enough to circumvent cellular barriers and are remarkably biocompatible. Reactive oxygen species (ROS) production is largely responsible for the toxicity that nanoparticles cause to cancer cells. Overproduction of ROS can lead to oxidative stress, disruption of normal physiological processes, and oxidation regulation. Thus, these effects may result in cytotoxicity, programmed cell death, damage to DNA, changes to cell signaling pathways, changes to cell development, and the onset of cell death [47,48]. Plant phytochemicals or nanoparticles coated with biopolymers have been used in medical therapies in many research studies [49]. Au NPs have demonstrated exceptional bioactivities, including antifungal, antioxidant, and antibacterial efficacy on a variety of bacteria and fungi, among other metal variations. Additionally, in vitro and in vivo research has thoroughly examined their potential as non-traditional chemotherapeutic medications and in the diagnosis of cancer [50]. We investigated the GO-CS/Au nanocomposite material to examine its antioxidant capability after the DPPH experiment and its cytotoxicity against human bladder cancer cells, such as 5637 and TCCSUP, after an MTT colorimetric assays.

2. Materials and Methods

2.1. Preparation of GO-CS/Au NPs

Using sonication, 200 mg of the synthesized GO was first suspended in 200 mL of deionized water. Dissolving 30 mg of chitosan in 50 mL of 1% acetic acid produced a chitosan solution separately. Next, while aggressively swirling the mixture, the chitosan solution was added dropwise to the GO dispersion. A 1 mL aqueous solution of glutaraldehyde (25 wt%) was then gradually added to the mixture while stirring for a further 4 h. The resulting composite solid was separated, cleaned as in the previous stage, and then dried for 12 h at 60°C to produce GO-CS, a dark-brown solid.

After that, 0.1 g of the GO-CS nanocomposite was dissolved in 100 mL of DI water and left to stir for 20 min. To evenly distribute the gold ions onto the nanocomposite, a solution of 10 mL chloroauric acid (HAuCl₄) (0.01 g/10 mL) was then added gradually to the mixture and agitated for 10 min. The mixture was then agitated for 1 h after adding 2 mL of pomegranate juice as a reducing agent. Following centrifugation, the resultant GO-CS/Au NPs underwent three water washes to remove any remaining materials before being vacuum-dried. Using ICP analysis, the catalyst’s gold concentration was determined to be 0.131 mmol g-1.

2.2. Typical reduction of nitroarenes by GO-CS/Au NPs

The catalytic reduction of nitrobenzene was examined by mixing 5 mL of 4-NP solution (2.5 mM) with a solution containing GO-CS/Au NPs (4.0 mg). After that, the mixture was rapidly agitated and 0.5 mL of a 25 mM NaBH4 solution was added. The lowering of 4-NP caused the solution’s yellow hue to progressively fade. The reaction progress was monitored via UV-Vis time dependent spectroscopic analysis. The catalyst underwent recovery by centrifugation following the concentration of the reaction filtrate to get the pure reduced product.

2.3. Antioxidant activities of GO-CS/Au nanocomposite

The antioxidant activity of a pharmaceutical drug or biological specimen is typically assessed using the DPPH free radical scavenging approach. At the outset, a DPPH solution was prepared by dissolving it in EtOH (0.002 g in 40 mL of 96% EtOH). Specifically, GO-CS/Au NPs nanocomposite suspensions (5 mL) at varying loads (1-1000 μg mL-1) were combined with DPPH solution (0.2 mM, 1 mL) the mixtures were kept in dark at 37°C for 60 min. The purple DPPH solution gradually disappeared after 30 min of incubation, causing in a shift in absorbance at 517 nm. To determine the % inhibition or DPPH scavenging capabilities, the given equation (1) was applied.

(1)
I n h i b i t i o n   r a t i o   ( % ) = A b s   o f   c o n t r o l − A b s   o f   s a m p l e A b s   o f   c o n t r o l × 100

2.4. Anti-bladder cancer potential of GO-CS/Au nanocomposite

The GO-CS/Au NPs nanocomposite’s cytotoxic investigations and anti-bladder cancer effects were assessed using the widely used an MTT colorimetric assay over 5637 and TCCSUP cell lines. The cancer cells were first cultivated in a humidified incubator with a CO2 environment at 37°C and roswell park memorial institute (RPMI)-1640 media containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic to maximize their ability to proliferate. The media was taken out of the culture vessel after it had been seeded with around 85% confluency. 0.5 mL of trypsin was applied to the cells after they had been cleaned with phosphate buffered saline (PBS), and they were then left for 5 min. The cells were taken out of the incubator and examined under an inverted microscope. RPMI-1640 (0.2 mL) growth media was used in each well to seed at a density of 1×105 cells/well in 96-well plates. The cells received varying dosages of GO-CS/Au NPs following a 24 h period of rest. MTT solution was then employed in combination with FBS media (10 μL). MTT reagent can pass through living cells’ cell membranes and the inner mitochondrial membrane due to its lipophilic nature and positive charge. Later, cells that are metabolically active decrease it to formazan. Due to its aqueous insolubility, the formazan is subsequently dissolved in dimethyl sulfoxide (DMSO) to afford a purple-colored solution. Subsequently, absorbance of the mixture was recorded using a microtiter plate reader set at 570 nm. To determine the % cell viability, the following equation (2) was used.

(2)
C e l l   v i a b i l i t y   ( % ) = T o t a l   c e l l s − D e a d   c e l l s T o t a l   c e l l s × 100

2.5. Statistical analysis

Statistical analysis was carried out in triplication and the outcomes were assessed using the one-way analysis of variance (ANOVA) and Least significant difference (LSD) test. p ≤ 0.05 was considered as a significant level.

3. Results and Discussion

3.1. Characterization data analysis of GO@CS/Au nanocomposite

To enclose the GO surface and impregnate it with Au3⁺ ions, the CS biomolecule, a polysaccharide with many amine and hydroxyl organofunctions was utilized. The coordination of entering Au3⁺ ions is facilitated by the amino and hydroxyl functionalities, which then lead to the sustained reduction of the pomegranate phytomolecule ions into NPs. Additionally, the coordination of the as-synthesized Au NPs with electron-rich oxygenous organic functions ensured stability (Scheme 1). Several state-of-the-art methods, including FE-SEM, EDX, elemental mapping, TEM, and XRD, were employed to thoroughly investigate the material’s physicochemical characteristics (Scheme 1).

Synthetic strategy of GO-CS/Au NPs and catalytic exploration in the reduction of nitrobenzene and anti-cancer application.
Scheme 1.
Synthetic strategy of GO-CS/Au NPs and catalytic exploration in the reduction of nitrobenzene and anti-cancer application.

FE-SEM analysis was first conducted to determine the shape, texture, and particle size of the GO-CS/Au NPs bio-nanomaterial, as displayed in Figure 1. The particles are in the nanometric range and have a definite spherical shape. The average particle size was found to be between 30 and 40 nm. Nevertheless, the image did not allow for the independent identification of the CS-GO composite or the Au NP deposition. With minimal aggregation, the particles seem to have distinct identities.

FE-SEM images of GO-CS/Au NPs at different magnifications (a) 10 μm and (b) 200 nm.
Figure 1.
FE-SEM images of GO-CS/Au NPs at different magnifications (a) 10 μm and (b) 200 nm.

Following the morphological analysis with field emission scanning electron microsocpy (FE-SEM), an elemental analysis with EDX was carried out. As shown in Figure 2, the GO-CS/Au bio-nanomaterial confirms the occurrence of Au, C, and O as constituent constituents. Perhaps because of its decreased concentration, N of CS did not occur. The non-metal C and O identification validates the CS-GO biocomposite. The map sum spectrum with the matching percentage weight of the constituent elements is shown inset. Au species (74.4%) account for the largest contribution, followed by other components, according to the related map sum spectrum (inset).

EDX pattern of GO-CS/Au NPs.
Figure 2.
EDX pattern of GO-CS/Au NPs.

TEM examinations were used to investigate the intrinsic structure of the GO-CS/Au bio-nanocomposite in greater detail. The results have been displayed in Figure 3 at different magnifications. The pictures depict the transparent structure that represents the GO surface, which resembles a sheet of butter paper. The different hues over GO might be a sign that CS and pomegranate phytomolecules have modified the surface. The spherical, black particles that are widely separated and do not appear to be aggregating are the clearest indication of Au NPs. Most of the particles are spherical in shape and between 30 and 35 nm in size.

TEM microstructures of GO-CS/Au nanocomposites at different magnifications (a) 250 nm, (b) 80 nm.
Figure 3.
TEM microstructures of GO-CS/Au nanocomposites at different magnifications (a) 250 nm, (b) 80 nm.

Additionally, the average size of gold nanoparticles over the surface of graphene oxide was 34.1 nm determined by particle size distribution diagram through TEM image (Figure 4).

Particle size histogram of GO-CS/Au NPs.
Figure 4.
Particle size histogram of GO-CS/Au NPs.

The architecture of the final material is explained by comparing the Fourier transform infra-red (FT-IR) profiles of GO-CS/Au and its precursors GO, extract, and GO-CS in Figure 5. The C–OH stretching vibration, broad O–H linked vibrations, and intercalated water stretching vibrations are responsible for the enlarged broad peak in the GO spectrum, which is located between 2900 and 3500 cm-1. Other distinct vibrations, which correspond to C-C, C=O, C=C, carboxyl O–H, and epoxy C–O stretching vibrations, are also detected at 2926, 1735, 1622, 1391, and 1110 cm-1 (Figure 5a). The spectrum of pomegranate extract, displayed in Figure 5b, shows a broad peak around 3000–3400 cm-1 corresponding to polyphenolic moieties. The alkene functions present therein were detected at 1631 cm-1. The GO-CS spectrum combined nanocomposite shown in Figure 5c retains all the spectral features of GO and additionally includes the specific peaks of chitosan, such as N–H bending of acetylated amine at 1604 cm-1, C–N stretching vibration of C–NH2 group at 1072 cm-1. Finally, The FT-IR spectra of the final GO-CS/Au nanocomposite depict all the peaks of GO-CS with shifting of peaks in slightly higher or lower regions, attributed to the coordination of Au NPs.

FT-IR spectra of (a) GO, (b) extract, (c) GO-CS, and (d) of GO-CS/Au NPs.
Figure 5.
FT-IR spectra of (a) GO, (b) extract, (c) GO-CS, and (d) of GO-CS/Au NPs.

The elemental examination was further extended by the mapping analysis, or energy-dispersive X-ray (EDX), which helps to comprehend the structural components and their distribution throughout the surface. The distribution of the corresponding atomic species is depicted by the colored dots that emerged by scanning a segment of the SEM image over X-ray (Figure 6). The EDX results are accompanied by the elemental data. It demonstrates how evenly distributed the atomic species of Au, C, and O are across the catalyst surface, which undoubtedly contributes significantly to catalysis.

Elemental mapping of GO-CS/Au NPs.
Figure 6.
Elemental mapping of GO-CS/Au NPs.

By using XRD analysis, the GO-CS/Au bio-nanocomposite’s crystallinity, purity, and phase composition were also determined. The profile is identical in Figure 7. The XRD profile shows a prominent diffraction peak at 25.8°, which corresponds to the (002) plane of reduced GO. Nevertheless, the profile also shows additional distinct diffraction signals at 2θ = 39.1°, 44.3°, 65.7°, and 78.4°, demonstrating the planes (111), (200), (220), and (311) of fcc Au crystals. Scherrer’s formula (D = Kλ/βcosθ) was used to calculate the crystallite size using FWHM data, and it was found the crystallite dimension is 33.25, and the data is close to the TEM analysis.

XRD pattern of GO-CS/Au NPs.
Figure 7.
XRD pattern of GO-CS/Au NPs.

3.2. Catalytic evaluation of GO-CS/Au NPs

A critical stage in many homogeneous and heterogeneous systems is the electron transfer step. A substantial discrepancy in the acceptor and donor’s redox potential may hinder electron transfer at this stage. Electron mobility is facilitated by an effective catalyst that serves as an electron relay system and has an intermediate redox potential value between the donor and acceptor partners. Metal NPs are commonly used as redox catalysts of this type. The reduction of 4-NP to 4-AP was thus used to evaluate the redox catalytic performance of GO-CS/Au NPs. When the substrate 4-NP and NaBH4 were first combined, the production of the 4-nitrophenoxide ion caused the solution to turn dark yellow. Electrons were then transferred to the substrate to initiate the redox reduction as soon as the catalyst was added. The solution lost its hue and became colorless after the 4-NP was entirely converted to 4-AP. To justify the role of Au in the reduction, a nanocomposite GO-CS was used instead of the main catalyst in the presence of excess NaBH4, but it was unable to carry out the reaction. CS only had the role towards biogenic green reduction of metal ions and subsequent stabilization of Au NPs. Figure 8 shows the observations that were tracked using UV-Vis spectroscopy. In the diagram, the second bell-shaped hump is caused by the substrate’s nitro group, which had a λmax of 400 nm. The full conversion of nitro to amines caused it to gradually smooth down over time.

Spectroscopy reduction of 4-NP using 5.0 mg GO-CS/Au NPs catalyst in the presence of NaBH4.
Figure 8.
Spectroscopy reduction of 4-NP using 5.0 mg GO-CS/Au NPs catalyst in the presence of NaBH4.

An array of nitro aromatics was reduced over the GO-CS/Au NPs catalyst under the same standard conditions, and the outcomes have been documented in Table 1. It was discovered that several substrate types, such as OH, OCH3, Cl, Br, NH2, CN etc., were equally compatible under the reaction conditions. Interestingly, electron-rich substrates like OH, CH3, OCH3, NH2, halogens, etc., at 2 or 4 positions enhance the electron density to the NO2 center which in turn facilitates the electronic reduction over the catalyst, and that has been reflected in the outcomes (entries 2, 3, 4, 9-11, etc). Conversely, the reactions of electron-withdrawing substrates were sluggish (entries 8, 12). Steric hindrance plays a role in the impact between the 2- and 4-methyl substituted nitrobenzenes where the 2-susbtrates are slightly less reactive (entries 10, 11). Excellent yields between 88 and 96 % were obtained from all reactions, which were finished in 0.5 to 3.0 h.

Table 1. Catalytic reduction of nitroarenes by GO-CS/Au NPs nanocatalyst.a
Entry Nitroarene Time (h) Yieldb (%) TOF (h-1)c
1 Nitrobenzene 1.5 95 974.3
2 4-Nitrophenol 0.5 96 2953.8
3 4-Nitroanisole 1.5 95 974.3
4 4-Choloro-nitrobenzene 1.0 90 1384.6
5 4-Bromo-nitrobenzene 1.5 92 953.5
6 4-Iodo-nitrobenzene 1.5 93 953.8
7 4-Nitroaniline 2.0 95 730.7
8 3-Choloro-nitrobenzene 2.5 92 566.1
9 2-Nitrophenol 2.0 90 692.3
10 4-nitrotoluene 1.0 93 1430.7
11 2-nitrotoluene 2.5 90 553.8
12 4-cyano nitrobenzene 3.0 88 451.2
aReaction condition: nitroarenes (1.0 mmol), NaBH4 (3 mmol), water (3 mL), GO-CS/Au NPs (5 mg, 0.065 mol%).
bIsolated yields.
cTOF = (yield/time)/amount of catalyst (mol).

Examining the reusability of this heterogeneous catalyst is practically necessary. Thus, centrifugation was used to recover the nanocatalyst following the completion of a new set of probe reactions. The residue was vacuum-dried at 60°C to regenerate after being properly cleaned with aqueous acetone. 4 mg of catalyst was employed in this experiment because the batch size was larger (2 mmol scale). Up to its seventh run, the GO-CS/Au nanocatalyst showed very good catalytic competence after being used in later batches (Figure 9a). Due to the catalytic material’s feeble leaching into the solution, there was minimal loss seen following the seventh batch. The TEM image of the recovered catalyst after the 7th cycle verifies that its shape stayed nearly the same (Figure 9b).

(a) Study of recyclability of the GO-CS/Au NPs in the reduction of 4-NP, and (b) The TEM image of recovered catalyst after the 7th cycle.
Figure 9.
(a) Study of recyclability of the GO-CS/Au NPs in the reduction of 4-NP, and (b) The TEM image of recovered catalyst after the 7th cycle.

To investigate the robustness of the material, leaching experiments were conducted. There was a trace quantity of Au leaching (<0.002 mmol g-1), based on the findings of the inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis of the filtrate from the 7th run. This filtrate was investigated with fresh substrates but found catalytically inactive. Following the hot filtering test, another investigation was conducted to determine the catalyst’s true heterogeneity. After the catalyst reached the midway point (15 min, 67%), it was centrifuged to recover and allowed to continue the reaction in that state for another 30 min. The reaction yield was remarkably heterogeneous, showing no further development.

3.3. Investigation of antioxidant potency and cytotoxicity of GO-CS/Au nanocomposite

Numerous investigations have demonstrated the exceptional antioxidant properties of Au nanocomposites, studied in vitro. Antioxidant substances related to Au NPs are highly desirable in the field of health management due to their ability to combat oxidative stress. Several studies have demonstrated the strong antioxidant potential of materials with very good anti-tumor properties and the capacity to cause cell death in malignancy. The radical was eventually quenched, turning the purple color to a pale yellow. The UV absorbance of the material was then measured to determine its antioxidant capacity. Figure 10a demonstrates the gradual increase in % inhibition with increasing concentration of the experimental antioxidant, GO-CS/Au nanocomposite. The IC50 value of the nanocomposite against DPPH free radicals was found to be 98 µg mL-1.

(a) Antioxidant analysis of GO-CS/Au NPs, and In vitro toxicity study of GO-CS/Au NPs on (b) 5637 cell and (c) TCCSUP cell.
Figure 10.
(a) Antioxidant analysis of GO-CS/Au NPs, and In vitro toxicity study of GO-CS/Au NPs on (b) 5637 cell and (c) TCCSUP cell.

After a significant antioxidant capacity was noted in an MTT experiment, the GO-CS/Au nanocomposite was then tested for cytotoxicity against the bladder cancer cell lines (5637 and TCCSUP). Because of the obvious linear relationship between absorbance and cellular activity, the MTT test, which measures the rate of cell growth, may detect cell proliferation with great sensitivity and accuracy. It is standard procedure to perform the MTT assay following a few hours of cell exposure to MTT, and the subsequent procedure follows, as mentioned in section 2.4.

According to the study, mitochondrial damage resulted from the GO-CS/Au nanocomposite’s considerable reduction in ATP levels in the cell. Additionally, the high ability of Au NPs to generate ROS makes them more cytotoxic. The dead cells in our research did not create formazan when the GO-CS/Au nanocomposite was introduced to the growing cells because they were devoid of this enzyme, which prevents them from becoming colorless. The percentage of cell viability was determined using equation 2 following the completion of the spectrometric tests. The cytotoxic activities of the nanomaterial on both cell lines demonstrate that when the amount of the cytotoxic material (GO-CS/Au nanocomposite) increases, the % toxicity increases. (Figure 10b, c). Based on the associated IC50 values from the cancer tests, 50% of the malignant cells in the 5637 and TCCSUP cell lines had to be destroyed by 648 and 458 µg mL-1 of the chemical, respectively. The cell line with the lowest IC50 value, TCCSUP, produced the best results, as per the data. Our research markedly displays that GO-CS/Au nanocomposite significantly inhibit the growth of human bladder cancer cells. Its effectiveness against malignant bladder cells is adequately demonstrated by the obvious lack of cell damage.

4. Conclusions

This study effectively demonstrated the efficiency of the GO-CS/Au nanocomposite by using phytochemicals that are secondary metabolites rather than pomegranate extract as a green reductant. Au NPs were synthesized green-metrically and then, in accordance with a green process, embedded over the high-surface GO. The nanocomposite’s effective synthesis and key characteristics were established through characterization utilizing FE-SEM, TEM, EDX, elemental mapping, and XRD. According to a TEM examination, the CS-functionalized GO surface has microscopic Au NPs with diameters of 30–35 nm embedded across it without any agglomerations. Additionally, XRD analysis indicated a crystalline, high-purity material that was supported by standards. The material was investigated for its potential use in chemical catalysis in the reduction of nitroarenes, a deadly water contaminant. In relatively little time and with exceptional yields, a variety of nitroarenes were reduced in aqueous media with NaBH4. Without a noticeable drop in activity, the catalyst was recovered by centrifugation and used for seven more trials. In addition, GO-CS/Au nanocomposite’s cytotoxic qualities were investigated against bladder cancer cell lines (5637 and TCCSUP) to conduct a biological investigation. The material’s matching IC50 was determined to be 648 and 458 µg mL-1, respectively, based on an MTT assay results. Using the DPPH assay, the substance was once more found to be a strong antioxidant, with a relative IC50 value of 98 µg mL-1. Due to infrastructural limitations, we could not determine the Trolox equivalent comparison and include the full inhibition curve. Also, we could not provide the mechanistic speculation about ROS and apoptosis following assays like 2′,7′-dichlorodihydrofluorescein diacetate reactive oxygen species (DCFH DA ROS), Annexin V PI, caspase activity, mitochondrial membrane potential, or western blot for apoptotic markers. In future works, as an extension, we will try to compensate for these shortcomings. With these outcomes we will further plan to continue our research in this nanomaterial domain on the catalytic applications of some complex reactions and in vivo biological studies on animals like rats.

Acknowledgement

The authors are thankful to Shanxi Province Cancer Hospital for providing necessary technical assistance.

CRediT authorship contribution statement

C. Zhang: Conceptualization and writing original text, K. Wu and T. Xing: Formal analysis, investigation and software, W. Wang: Project administration, editing original text and review.

Declaration of competing interest

The authors declare there is no competing interest.

Data availability

The data will be available on request to authors.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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