Design and synthesis of chitosan/calcium lignosulfonate/Au NPs: Its performance for reduction of nitro compounds and in the treatment of cancer

2.1 Synthesis of the CLS-CS/Au NPs

First, equal proportions of chitosan (CS) and calcium lignosulfonate (CLS) (0.1 g each) were dissolved in 1 % acetic acid solution (50 mL) through stirring for 60 min at 25 °C to make a homogenous CLS-CS composite. Next, the pH of the reaction medium was adjusted to pH 10 and a 10 mL aqueous solution containing 0.02 g HAuCl₄ was added dropwise to the above mixture and stirred for 5 h at 80 °C. Finally, the obtained CLS-CS/Au NPs nanocomposite were collected by centrifuge and rinsed with deionized water and ethanol sequentially.

2.2 General reduction of nitroarenes catalyzed by CLS-CS/Au NPs

For this purpose, nitroarenes (1 mmol) and NaBH₄ (3 mmol) was added to deionized water (5 mL) and stirred to obtain a homogeneous solution. Next, the CLS-CS/Au NPs nanocomposite (0.2 mol% Au) was added to the solution, and the reaction was allowed to proceed for desired time at 25 °C. End of the reaction, the catalyst was separated over centrifuge and the organic layer was dried over dehydrated Na₂SO₄ and the crude products were purified by using a pad column chromatography.

2.3 Antioxidant activities of CLS-CS/Au NPs nanocomposite

In this approach, varying concentrations of nanoparticles (ranging from 0 to 1000 µg/ml) were mixed with 1 mL of DPPH (300 µmol/l). The resulting mixture was then diluted with methanol to reach a final volume of 4000 µl. Subsequently, the samples were incubated in darkness for 1 h. The absorbance at 517 nm was measured. The DPPH radical inhibition percentage was determined by the equation (Lu et al., 2021). $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

2.4 Anti-pancreatic carcinoma, anti-gastric cancer, and anti-colorectal cancer properties of CLS-CS/Au NPs

HT-29 (colorectal carcinoma cell), MKN45 (Gastric cancer cell), and PANC-1 (Pancreatic cancer cell) were used in the current research.

Cell culture involves the cultivation of eukaryotic cells in a specialized medium that provides optimal growth conditions for each cell type. To support their growth, artificial environments typically include essential nutrients like gases, hormones, amino acids, sugars, minerals, and vitamins. Additionally, the chemical and physical conditions of the environment must be carefully regulated to promote cell growth. Depending on the cell type, some cells require a surface to attach to in order to grow, while others can thrive while floating in the culture medium. In laboratory settings, specific cell culture mediums are designed to cater to the needs of particular cell types (Abbasi et al., 2021; Ma et al., 2020). To ensure optimal cell proliferation, it is important to maintain a moderate cell density in the culture medium. Regular transfer of cells to fresh medium, known as passage, should be performed every few days. If the cell volume and density raise too much, it can hinder cell proliferation and lead to cellular differentiation. The objectives of cell culture include evaluating cell growth, nutritional needs, growth arrest, and observing changes in cell morphology under a microscope. In order to investigate the cell growth cycle, control the cancer cells growth, and examine gene expression, it need to conduct cell culture outside of the organism. This allows for a more detailed analysis of the specific type of growth and cells growth rate. Cell culture also finds application in the study of animal evolution. By culturing a fertilized cell in a laboratory setting, we can explore how it develops into a multicellular organism and observe the morphological differences of each cell under a microscope. This approach enables us to understand the cell culture stages in relation to evolution (Lu et al., 2021). Within the cell culture medium, cells are prepared and undergo differentiation, giving rise to new cells with the aid of growth factors and hormones. Through the cell culture process, cells with identical characteristics are generated, allowing for the study of various intracellular functions and cell metabolism. Once a molecule binds to its specific membrane receptor, subsequent intracellular reactions including the formation of complexes, intracellular messaging, and message transmission can be assessed. These cultured cells have the advantage of being able to be stored at extremely low temperatures, which helps to preserve their genetic composition and growth rate. Consequently, these cells can be utilized whenever necessary, even if it is a month later. Cell aging can be effectively prevented using this approach. When conducting studies involving animals like rats and rabbits, it is crucial to take into account animal homeostasis and experimental stress. However, when it comes to cell culture, these factors do not apply. The standardization of laboratory tests is simpler compared to studying animals, as it allows for better control over cell environments, including osmotic pressure, heat, acidity, as well as carbon dioxide and oxygen pressures (Lu et al., 2021).

In the recent study, the cells were stored in an incubator with 5 % CO₂ at 37 °C. Once the flask was approximately 80 % filled, cell passage was conducted, and around 10⁵ cells were seeded in 24-well petri dishes under normal environmental conditions. After 24 h, the cells were treated with nanoparticles. The cultured cells' survival rate was assessed using various concentrations. For this study, cells were cultured at a density of 3 × 10⁴ cells/well in 24-well plates and placed in an incubator at 37 °C. Subsequently, the old culture medium was aspirated from the wells, and the cells were exposed to varying nanoxidro concentrations. This assessment was conducted on the 1th, 2th, and 3th days post-treatment; hence, after the appropriate incubation period in the 24-well plates, the culture medium was replaced with approximately 300 μl of new medium in each well. After an incubation period of 3–4 h at 37° C, the MTT solution is aspirated and 200 μl of DMSO from Merck, USA (100 %) is added to each well. Subsequently, the samples absorbance was measured at a wavelength of 570 nm by an ELISA reader. This procedure was replicated thrice, with four wells allocated for each concentration of nano oxide. The cell viability percentage was determined using the formula (Lu et al., 2021): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

2.5 Statistical analysis

The Mean ± SE was calculated for the results by SPSS-22. Statistical tests of variance were conducted using a completely randomized block design. Graphs were created in Excel software (P < 0.05).

3.1 Structural study of the synthesized CLS-CS/Au NPs

The preparation of CLS-CS/Au NPs nanocomposite was described by a post-synthetic modification method based on CS and CLS, which used for in situ fabrication of Au NPs. The final CLS-CS/Au NPs nanocomposite was subsequently analyzed using a number of advanced techniques like FT-IR, TEM, FE-SEM, EDX-elemental mapping and ICP-OES.

To confirm the successful fabrication of CLS-CS/Au NPs nanocomposite, FT-IR analysis is recorded and presented in Fig. 1. The representative peaks of chitosan are appeared at 619, 1030–1050, 1151, 1382, 1543, 1600 and 2854–2924 cm⁻¹ assigned to N—H, C—O, C—O—C, C—N, in plane N—H bending, C⚌O stretching and C—H stretching respectively. The broad peak observed at 3431 cm⁻¹ assigned to the mixed N—H and O—H stretching vibrations. Also the presence of CLS could be confirmed with appearance the typical peaks for SO₃ adsorption at 673 cm⁻¹, C—O stretching at 1151 cm^-1and O—H stretching at 3400–3500 cm⁻¹. These observations shown the well attachment of Au NPs to the CLS-CS matrix.

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Fig. 1

FT-IR spectrum of CLS-CS/Au NPs.

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The microscopic structures, morphology and size distributions of the CLS-CS/Au NPs were determined by FE-SEM and TEM studies. Fig. 2 shown the FE-SEM analysis of the CLS-CS/Au NPs composite, which indicating the successful preparation of spherical Au NPs that encapsulated in the CLS-CS hydrogel layers. For further detailing regarding the particle size and their distribution, TEM analysis was performed (Fig. 3). The result presented spherical Au NPs as dark spots with an average size of 20–30 nm.

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Fig. 2

FE-SEM image of the CLS-CS/Au NPs.

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Fig. 3

TEM image of the CLS-CS/Au NPs.

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Fig. 4 presented the profile for chemical constitution of CLS-CS/Au NPs, as analyzed by EDX investigations, equipped with SEM instrument. It clearly shows Au as metallic elements as a major and broad signal at 2.1 eV. The non-metallic signals of C and O appeared at lower region are attributed to the CLS-CS functionalization coated over the surface. Next, an elemental mapping study was conducted to further extend the elemental analysis. (Fig. 5). The obtained results shows the constitutional elements and their respective positions as represented by colored dots scattered over the material surface.

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Fig. 4

EDX of the CLS-CS/Au NPs.

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Fig. 5

Elemental mapping of CLS-CS/Au NPs.

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3.2 Reduction of nitroarenes catalyzed by CLS-CS/Au NPs

After the characterization of CLS-CS/Au NPs, its catalytic performance was investigated for reduction of nitroarenes. So, to optimize the reaction conditions by choosing a model reduction involving 4-nitrophenol and the effect of various parameters such as amount catalyst and amount of NaBH₄ were studied that presented in the Table 1. The reaction did not proceed in the absence of the catalyst, even after the use of NaBH₄ (3.0 mmol) for long time (Table 1, entry 1). However, the reaction was good using CLS-CS/Au NPs as catalyst and the best result (96 % yield) was achieved in 10 min when 0.2 mol% Au composite was applied in the presence of NaBH₄ (3.0 mmol) (Table 1, entry 6). We experimented with different amount of catalyst and NaBH₄ (Table 1, entries 7–9), but could not obtained better results than the entry 6.

Table 1 Evaluation of reaction parameters in the reduction of 4-Nitrophenol catalyzed by CLS-CS/Au NPs.^a

Entry	Catalyst	Cat. Loading (Au mol%)	NaBH4 (mmol)	Time (min)	Yield (%)b
1	–	–	3.0	180	–
2	CS	–	3.0	180	–
3	CLS-CS	–	3.0	180	–
4	CLS-CS/Au NPs	0.20	1.0	30	75
5	CLS-CS/Au NPs	0.20	2.0	20	80
6	CLS-CS/Au NPs	0.20	3.0	10	96
7	CLS-CS/Au NPs	0.20	4.0	10	96
8	CLS-CS/Au NPs	0.30	3.0	8	96
9	CLS-CS/Au NPs	0.10	3.0	30	85

aCondition: 4-nitrophenol (1.0 mmol) in 5 mL H₂O at r.t.

bIsolated yield.

After obtain the optimized conditions, its generality was investigated with a wide variety of nitroarenes having different functions (Table 2). Both the withdrawing and electron-donating groups were resulting excellent yields under the optimized protocol in short times (Table 2, entries 1–10).

Table 2 Generality of reduction in the presence of CLS-CS/Au NPs catalyst.^a

Entry	Substrate	Product	Time (min)	Yield (%)b
1			40	96
2			15	96
3			15	96
4			20	95
5			20	96
6			15	96
7			20	95
8			30	92
9			60	90
10			60	92

aReaction condition: nitroarenes (1.0 mmol), NaBH₄ (3 mmol), water (5 mL), CLS-CS/Au NPs (0.2 mol%).

bIsolated yields.

The reusability of the catalyst was investigated by model reaction and shown it could reuse 8 runs without significant loss of its catalytic activity (Fig. 6).

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Fig. 6

Study of reusability of CLS-CS/Au NPs.

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3.3 Antioxidant effects analysis of CLS-CS/Au NPs

Metallic nanoparticles consist of various compounds, each possessing a unique structure. The compounds extraction process is influenced by various factors, with the type of extraction and solvent method being the most crucial. Selecting a suitable solvent for a specific group of metallic nanoparticles compounds can be challenging due to the presence of other substances that impact the solubility of these compounds. When extracting NPs, it is essential to utilize a method that ensures optimal preservation of antioxidant compounds. Typically, metallic nanoparticles possess distinct antioxidant properties. Recent research has indicated that when these NPs are incorporated into metal nanoparticles as reducing and stabilizing agents, they create nanocomposites with exceptional antioxidant efficacies. Antioxidants are commonly known as substances capable of delaying, decelerating, and even halting oxidation processes. They effectively safeguard against alterations in the color and food flavor caused by oxidation reactions (Beheshtkhoo et al., 2018; Sangami and Manu, 2017; Katata-Seru et al., 2018; Sankar et al., 2014). The mechanism of oxidants can be counteracted by their ability to hinder the propagation of oxidation chain reactions through the provision of hydrogen atoms to free radicals. However, the synthetic antioxidants utilization like TBHQ, BHA, BHT, and other chemical additives has been restricted in recent times due to their potential toxicity and carcinogenic properties. Presently, the primary emphasis in research within this field lies in exploring the application of novel and harmless antioxidants derived from food, microbial, animal, and plant sources (Conde et al., 2012; Riehemann et al., 2009; Rai et al., 2014; Jo et al., 2015; Bhattacharya, 2011).

Various drugs are made using metallic nanoparticles secondary metabolites. These metabolites include amino acids, flavonoids, phenols, saponins, steroids, terpenoids, and alkaloids. The production of these drugs has been successful, thanks to the presence of these metabolites (Beheshtkhoo et al., 2018; Sangami and Manu, 2017). Flavonoids, which are part of the large family of secondary metabolites, have a structural skeleton of diphenylpropane or polyphenols and including two aromatic benzene rings. Currently, there have been 6,000 different flavonoids identified, with major ones being compferrol, quercetin, and myristin. Luteolin and apigenin are the two primary flavonoids, encompassing a total of 62 distinct types that can be found in a range of fruits, vegetables, and edible tropical NPs. These flavonoids possess several well-known effects, including the ability to eliminate free radicals, inhibit certain enzymes, and exhibit anti-inflammatory effects (Beheshtkhoo et al., 2018; Sangami and Manu, 2017; Katata-Seru et al., 2018; Sankar et al., 2014). Some evidence suggests that the compounds biological impacts are linked to their antioxidant efficacy. Antioxidants function by several mechanisms within the body, such as exerting anti-inflammatory effects, inducing apoptosis, boosting the immune system, halting the cell cycle, and promoting cell differentiation (Beheshtkhoo et al., 2018; Sangami and Manu, 2017; Katata-Seru et al., 2018; Sankar et al., 2014; Namvar et al., 2014).

Table 3 and Fig. 7 present the findings of a recent study, highlighting the scavenging capacity of CLS-CS/Au NPs and BHT at various concentrations. The antioxidant test revealed that the IC50 values of BHT and CLS-CS/Au NPs against DPPH free radicals were 104 and 120 µg/mL, respectively, as shown in Table 3.

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Fig. 7

The antioxidant properties of BHT, CLS-CS/Au NPs, and HAuCl₄ against DPPH.

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3.4 Analysis of anti-colorectal carcinoma, anti-gastric cancer, and anti-pancreatic cancer properties of CLS-CS/Au NPs

The antioxidant effects of recent nanoparticles seem to contribute to their ability to inhibit esophageal cancer in humans. Given the strong connection between tumor progression, oxidative stress, and inflammation, compounds with antioxidant or anti-inflammatory properties can potentially act as agents to prevent cancer (Beheshtkhoo et al., 2018; Sangami and Manu, 2017). Numerous nanoparticles possess biochemical and pharmacological properties, such as anti-inflammatory and antioxidant effects, which play a role in their anticarcinogenic and antimutagenic activities (Katata-Seru et al., 2018; Sankar et al., 2014; Namvar et al., 2014; Chen and Schluesener, 2008; Beyene et al., 2017; Radini et al., 2018; Huang et al., 2017; Conde et al., 2012).

Cancer, known for its abnormal cell proliferation and uncontrolled growth, stands as a prominent contributor to mortality. Its development can be attributed to both external factors like chemicals and tobacco, and internal factors like hormones and genetic mutations. To combat this disease, a range of treatment methods including surgery, radiation therapy, and chemotherapy are employed. However, a major challenge lies in delivering targeted drugs to cancer cells without adversely affecting healthy cells, leading to high cancer mortality rates (Huang et al., 2017; Conde et al., 2012; Riehemann et al., 2009; Rai et al., 2014). Nanotechnology appears to offer a promising solution to this issue. The distinctive characteristics of nanoparticles, including their size, surface-to-volume ratio, targeting capabilities, drug-loading capacity for water-insoluble drugs, controlled release mechanisms, and responsiveness to stimuli, position them as viable candidates for cancer therapy (Rai et al., 2014; Jo et al., 2015; Bhattacharya, 2011; Alexander, 2009). When nanoparticles are introduced into the bloodstream, they must traverse the arterial walls to access the desired location and subsequently discharge the medication. Nanoparticles, being larger in size compared to small molecules, are unable to navigate through the narrow gaps between the normal blood vessels endothelial cells. Conversely, the permeable walls of tumor blood vessels facilitate the passage of nanoparticles. Targeted drug administration to the tumor using nanoparticles involves both active and passive targeting mechanisms. Passive targeting leverages the characteristics of the tumor tissue for drug delivery (Radini et al., 2018; Huang et al., 2017; Conde et al., 2012; Riehemann et al., 2009; Rai et al., 2014; Jo et al., 2015). Conversely, the tumor tissue lymphatic system is incomplete and unable to collect nanoparticles and transport them into the bloodstream. In order to achieve passive targeting, the nanoparticles size should generally be below 200 nm. On the other hand, tumor active targeting is accomplished by attaching a ligand such as aptamers, peptides, antibodies, or certain small molecules like folic acid to NPs (Riehemann et al., 2009; Rai et al., 2014; Jo et al., 2015; Bhattacharya, 2011; Alexander, 2009).

The MTT examination was conducted to check the anti-pancreatic cancer, anti-gastric cancer, anti-colorectal carcinoma, and cytotoxicity efficacies of the treated cells with CLS-CS/Au NPs over a 48-hour period on normal (HUVEC) and cancer cells (Fig. 8, 9).

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Fig. 8

The cytotoxicity effects of CLS-CS/Au NPs and HAuCl₄ on normal cell.

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Fig. 9

The anti-cancer properties of HAuCl₄ and CLS-CS/Au NPs against SK HT-29 (colorectal carcinoma cell), MKN45 (Gastric cancer cell), and PANC-1 (Pancreatic cancer cell).

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The IC₅₀ values of the CLS-CS/Au NPs were found to be 169 µg/mL for HT-29 (colorectal carcinoma cell), 196 µg/mL for MKN45 (Gastric cancer cell), and 112 µg/mL for PANC-1 (Pancreatic cancer cell). The growth of malignant colorectal, gastric and pancreatic cells was found to decrease in a dose-dependent manner when exposed to CLS-CS/Au NPs (Table 4; Fig. 8, 9).