Preparation of gelatin/Ag NPs under ultrasound condition: A potent and green bio-nanocomposite for the treatment of pleomorphic hepatocellular carcinoma, morris hepatoma, and novikoff hepatoma

2.1 Materials and methods

The chemicals required for catalyst synthesis and catalyst applications were purchased from Sigma Aldrich.

2.2 Synthesis of Ag NPs@gelatin bionanocomposite

To initiate the reaction 20 mL of a freshly prepared aqueous solution of AgNO₃ (10 mM) was added to 100 mL of an aqueous gelatin suspension under ultrasonic conditions and left for 10 min at 60 °C. Then the pH of the suspension was adjusted to 11.0 (NaOH, 3 wt%) and the reaction mixture was kept with sonication at 60 °C for 60 min. The progress of the reaction could be monitored by the change in color from watery yellow to brown color of the aqueous solution was due to the excitation of the surface plasmon resonance (Stapleton and Nurkiewicz, 2014) and SPR band which both play an important role in the confirmation of silver nanoparticles formation (Patra et al., 2018). The prepared Ag NPs@gelatin were collected by centrifugation and washed several times with DI-water. Ag content in the catalyst was 3.6 mmol/g, as determined by ICP-OES analysis.

2.3 Antioxidant activities of Ag NPs@gelatin bionanocomposite

α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method offers the first approach for evaluating the antioxidant potential of a compound, an extract or other biological sources. This is the simplest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded after a defined period. However, with the advancement and sophistication in instrumental techniques, the method has undergone various modifications to suit the requirements, even though the basic approach remains same in all of them (Lu et al., 2021).

In this method, 1 mL of different concentrations of the nanoparticles (0–1000 µg/ml) (with 1 mL of DPPH (300 µmol/l) combined and then the final volume of the combination with methanol reached 4000 µl. The falcons were then vertexed and kept in the dark for 60 min. The absorbance was read at 517 nm. The DPPH radical inhibition percentage was calculated using the following 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-liver carcinoma properties of Ag NPs@gelatin bionanocomposite

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. The lifespan of most cells is genetically determined, but some cell culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided. In practice, the term “cell culture” now refers to the culturing of cells derived from multicellular eukaryotes, especially animal cells, in contrast with other types of culture that also grow cells, such as plant tissue culture, fungal culture, and microbiological culture (of microbes). The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Viral culture is also related, with cells as hosts for the viruses. The laboratory technique of maintaining live cell lines (a population of cells descended from a single cell and containing the same genetic makeup) separated from their original tissue source became more robust in the middle 20th century (Lu et al., 2021).

In this study, common human liver cancer cell lines i.e. pleomorphic hepatocellular carcinoma (SNU-387), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) were used to evaluate the anticancer effect of Ag NPs@gelatin bionanocomposite on cell culture.

These cells in DMEM culture medium (Gibco, USA) with 10% FBS (Gibco, USA) and penicillin/streptomycin (100 μl/100 μg/ml) in an incubator containing 5 % Carbon dioxide with 90% humidity was stored at 37 °C. Then, when about 80% of the flask was filled, cell passage was performed and about 5 × 10⁴ cells (per square centimeter) were placed in 24 house bacterial petri dishes in the usual environment. The cells were treated with different concentrations of nanoparticles 24 h later and kept in this condition for 3 days. The survival rate of cultured cells was prepared with different concentrations. In this experiment, cells were cultured at 3 × 10⁴ cells/well in 24-well plates and kept in an incubator at 37 °C for 24 h. Then the old culture medium was taken out of the wells and the cells were treated with different concentrations of nanoxidro. This test was performed on the first, second and third days after exposing the cells to the compounds; thus, at the appropriate time after culturing the cells in plates of 24 cells, the culture medium was removed and about 300 μl of fresh medium containing 30 μl of MTT solution was added to each cell. After 3–4 h of incubation at 37 °C, MTT solution is removed and 200 μl (Dimethyl Sulfoxide, Merck, USA, 100%) DMSO is added to each house. Then the sample absorption was read at 570 wavelengths using ELISA rider (Expert 96, Asys Hitch, Ec Austria). This experiment was repeated 3 times and each time, four wells were considered for each nano oxide concentration. Cell survival percentage was evaluated by the following formula (Lu et al., 2021): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

The results were evaluated as Mean ± SE using SPSS software version 12 and statistical tests of variance of completely randomized block design. Drawing graphs in Excel software was performed and the significance level of the differences was considered p < 0.05.

3.1 Characterization of the prepared Ag NPs@gelatin bionanocomposite

The formation of Ag NPs in gelatin was established with UV–Visible spectrophotometer. The presence of specific peaks 440 nm confirmed the formation of Ag NPs as shown in Fig. 1. The broad peaks formed by Ag NPs in hydrogel is an indication of the reducing action of gelatin to silver nanoparticles. The successful biogenic synthesis of Ag NPs was primarily confirmed by a visual color change from watery yellow to brown color.

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Fig. 1

UV–visible absorption spectra of Ag NPs@gelatin bionanocomposite.

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The formation mechanism of Ag NPs@gelatin bionanocomposite and the attachment of gelatin can be explained on the basis of their FT-IR spectra (Fig. 2). The spectra of gelatin (Fig. 2a) showed vibration bands at 3420 cm⁻¹ (N—H stretch coupled with hydrogen bonding), 3088 cm⁻¹ (alkenyl C—H stretch), 2962 cm⁻¹ (CH₂ asymmetrical stretching), 1629 cm⁻¹ (C⚌O stretch/HB coupled with COO—), 1530 cm⁻¹ (N—H bend coupled with CN stretch), 1442 cm⁻¹ (CH₂ bend), 1241 cm⁻¹ (NH bend), and 1078 cm⁻¹ (C—O stretch) (Thi and Nguyen, 2016). Fig. 2b, showing the vibrational peaks of the Ag NPs@gelatin bionanocomposite, looks very similar to that of gelatin. This implies that gelatin is successfully encapsulated over the Ag NPs and there occurs no change in the internal core. Nevertheless, there occurs a slight relocation of the peaks in the spectrum of final material, which can be anticipated due to strong bond formation of Ag NPs with gelatin functions.

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Fig. 2

FT-IR spectrum of (a) gelatin and (b) Ag NPs@gelatin bionanocomposite.

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The surface morphology of the Ag NPs@gelatin bionanocomposite was investigated by the FE-SEM study, as shown in Fig. 3. It shown the quasi-spherical shaped nanoparticles of mean diameter of nano. For further structural inherence of the nanocomposite, TEM analysis was carried out (Fig. 4). The TEM measurements revealed the presence of spherically shaped nanoparticles in the matrix (Fig. 4). The mean diameter of Ag NPs was in the range of ∼5–10 nm. In the image, the nanoparticles were well dispersed without any aggregation.

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Fig. 3

FE-SEM image of the Ag NPs@gelatin bionanocomposite.

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Fig. 4

TEM images of the Ag NPs@gelatin bionanocomposite.

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The prepared Ag NPs@gelatin bionanocomposite was validated from EDX analysis, as presented in Fig. 5. The profile shows a major and distinct signal of Ag appeared at 2.95 keV. However, some other peaks of C, N and O as constitutional elements are also observed. They can be corresponded to the gelatin hydrogel, being associated with the Ag NPs surfaces. On X-ray scanning of a section of SEM image, the compositional map can be identified. It shows a uniform distribution of all the elements over the composite surface which is very significant for a bionanocomposite, particularly for the active species (Fig. 6).

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Fig. 5

EDX spectrum of the Ag NPs@gelatin bionanocomposite.

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Fig. 6

Elemental mapping of Ag NPs@gelatin bionanocomposite.

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The nature of crystalline phases of Ag NPs@gelatin bionanocomposite was investigated by XRD analysis, being shown in Fig. 7. While the initial phase in the 2θ region up to 15° was attributed to gelatin hydrogel and non-crystalline. Additionally, the diffraction peaks at 2θ = 38.1°, 44.3°, 64.6° and 77.2° confirms the Ag crystalline phases. These corresponds to the diffraction on fcc silver (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes (JCPDS No. 87-0717).

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Fig. 7

XRD pattern of the Ag NPs@gelatin bionanocomposite.

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3.2 Antioxidant properties of Ag NPs@gelatin bionanocomposite

In this study, we assessed the antioxidant properties of Ag NPs@gelatin bionanocomposite by using the DPPH test as a common free radical.

The scavenging capacity of Ag NPs@gelatin bionanocomposite and BHT at different concentrations expressed as percentage inhibition has been indicated in Fig. 8. In the antioxidant test, the IC50 of Ag NPs@gelatin bionanocomposite and BHT against DPPH free radicals were 203 and 250 µg/mL, respectively (Table 1 and Fig. 8).

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Fig. 8

The antioxidant properties of AgNO₃, Ag NPs@gelatin bionanocomposite, and BHT against DPPH.

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3.3 Anti-liver carcinoma effects analysis of Ag NPs@gelatin bionanocomposite

Unique properties of nanoparticles such as particle size, high surface-to-volume ratio, their targeting ability, loading ability of water-insoluble drugs and controlled release, and responsiveness to the stimulant drug can make them a good candidate for cancer treatment (Sankar et al., 2014; Beheshtkhoo et al., 2018; Radini et al., 2018; Katata-Seru et al., 2018; Sangami and Manu, 2017; Beyene et al., 2017; Chen and Schluesener, 2008; Alexander, 2009; Bhattacharya, 2011; Huang et al., 2017; Conde et al., 2012; Rai et al., 2014). When the nanoparticles are injected into the bloodstream, they need to pass through the walls of the arteries to reach the target site and then release the drug. Unlike small molecules, nanoparticles, because of their relatively large size, cannot pass through the tight connections between the endothelial cells of healthy blood vessels. Tumor blood vessels, due to their leaky walls, allow nanoparticles to pass well. Targeted drug delivery to the tumor by nanoparticles is done in both passive and active targeting (Rai et al., 2014; Jo et al., 2015; Riehemann et al., 2009; Bhattacharyya et al., 2011; Day et al., 2009; Pelaz et al., 2015). In passive targeting, tumor tissue properties are used for drug delivery. In tumor tissue, due to rapid cell growth, rapid angiogenesis occurs, but the distance between vascular endothelial cells is too large, causing macromolecules and nanoparticles to leak from blood vessels into tumor tissue (Pelaz et al., 2015; Sperling and Parak, 2010; Sau et al., 2010). On the other hand, the lymphatic system of tumor tissue is not complete and is not able to collect nanoparticles and insert them into the bloodstream; As a result, nanoparticles accumulate in the tumor tissue and release their drug. For passive targeting, the nanoparticle size should usually be below 200 nm. Active targeting of the tumor is achieved by binding a ligand such as antibodies, peptides, aptamers and some small molecules such as folic acid to nanoparticles (Riehemann et al., 2009; Bhattacharyya et al., 2011; Day et al., 2009; Pelaz et al., 2015; Sperling and Parak, 2010; Sau et al., 2010).

In this research, the treated cells with different concentrations of the present Ag NPs@gelatin bionanocomposite were assessed by MTT assay for 48 h about the cytotoxicity properties on normal (HUVEC) and human liver malignancy cell lines i.e. pleomorphic hepatocellular carcinoma (SNU-387), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) (Fig. 9,10).

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Fig. 9

The cytotoxicity effects of AgNO₃ and Ag NPs@gelatin bionanocomposite against normal cell line.

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The absorbance rate was evaluated at 570 nm, which represented viability on normal cell line (HUVEC) even up to 1000 μg/mL for Ag NPs@gelatin bionanocomposite (Table 2 and Fig. 9).

The viability of malignant liver cell line reduced dose-dependently in the presence of Ag NPs@gelatin bionanocomposite.

The IC50 of Ag NPs@gelatin bionanocomposite were 192, 144, and 228 µg/mL against pleomorphic hepatocellular carcinoma (SNU-387), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cell lines, respectively (Table 2 and Fig. 10).

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Fig. 10

The anti-human liver cancer properties of AgNO₃ and Ag NPs@gelatin bionanocomposite against liver cancer cell lines (a: pleomorphic hepatocellular carcinoma (SNU-387), b: morris hepatoma (McA-RH7777), c: novikoff hepatoma (N1-S1 Fudr)).

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