Green preparation of copper nanoparticle-loaded chitosan/alginate bio-composite: Investigation of its cytotoxicity, antioxidant and anti-human breast cancer properties

2.1 Preparation of the Cu NPs@CS/Alg nanocomposite

First, 0.5 g of chitosan (98% NH₂) and 0.5 g sodium alginate were dissolved in100 mL of 2% (v/v) acetic acid solution and sonicated for 30 min. Next it stirring at room temperature overnight to obtain chitosan/sodium alginate composite (CS/Alg). Then pH of the chitosan/alginate solution was adjusted to pH 10 (NaOH, 3 wt%) and an aqueous solution of CuSO₄·5H₂O (50 mg in 10 ml) is added drop-wise over 10 min, at room temperature. The mixture is mechanically stirred for 6 h at 100 °C. The progress of the reaction could be monitored by the change in color from watery blue to the dark-brown color of the aqueous solution was due to the excitation of the surface plasmon resonance and SPR band which both play an important role in the confirmation of copper nanoparticles formation. The prepared Cu NPs@CS/Alg nanocomposite were collected by centrifugation and washed several times with DI-water and dried.

2.2 Antioxidant activities of Cu NPs@CS/Alg nanocomposite

The free radical scavenging test was first performed by Blois in 1958, and after some modification by numerous studies in its current form. DPPH method is one of the most widely used methods for estimating antioxidant content. DPPH is a stable radical that reacts with hydrogen atom compounds. This test is based on the inhibition of DPPH, which causes the decolorization of DPPH solution by adding radical species or antioxidants. DPPH changes color from purple to yellow by taking an electron from the antioxidant compound. The free radicals in DPPH are adsorbed at 517 nm, which follows Beer Lambert's law, and decreased absorption is linearly related to the amount of antioxidants; the higher the amount of antioxidants, the more DPPH is consumed and the more purple turns yellow (Lu et al., 2021).

This experiment was performed with few changes in the method of Lu et al (Lu et al., 2021). 0.5 ml of 0.1 mM DPPH solution prepared in 95% ethanol was mixed with 100 μl of Cu NPs@CS/Alg nanocomposite. The resulting solution was kept in the dark at 38 °C for 31 min. The absorbance of the samples was then read at 518 nm (Lu et al., 2021).

To compare the activity of Cu NPs@CS/Alg nanocomposite, standard BHT compound was used as a standard antioxidant (Lu et al., 2021).

To determine the amount of IC50 (IC50 is defined as the concentration required to inhibit 50% of the antioxidant activity) for Cu NPs@CS/Alg nanocomposite, experiments were performed at eleven different concentrations of the desired nanoparticle solution and BHT. Each experiment was performed in three shifts and the mean values ​​were calculated (Lu et al., 2021).

Percentage of radicalization activity was calculated through the following equation (Lu et al., 2021): $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

In this regard, the blank adsorption indicates the adsorption of the control solution, which contains 0.5 ml of DMPH solution and 100 μl of 95% ethanol instead of Cu NPs@CS/Alg nanocomposite solution and adsorption of the reaction indicates the adsorption of the solution content of the Cu NPs@CS/Alg nanocomposite sample (Lu et al., 2021).

2.3 Anti-human breast cancer properties of Cu NPs@CS/Alg nanocomposite

One of the cytotoxicity test methods to measure the rate of cell death is the MTT method, which is based on the formation of formazan dye by reducing the substance MTT (dimethyl thiazole 2 and 5 diphenyltetrazolium bromide) or other tetrazolium salts (Lu et al., 2021). By breaking the MTT tetrazolium ring by mitochondrial enzymes in living cells, insoluble purple formazan crystals are formed. The formation of these crystals indicates the activity of respiratory chain enzymes and is a measure of cell viability. By measuring the amount of absorption by spectrophotometer at specific wavelengths, the number of living cells can be determined. This test is performed according to ISO 10993-5 and its purpose is in vitro evaluation of cytotoxicity. Cytotoxicity test is performed according to ISO10993-5 standard and in three ways: NRU test, CFU test, MTT test and XTT test. The most common method for assessing cytotoxicity is to measure cell survival by MTT (Lu et al., 2021). The basis of MTT method is based on the intensity of dye produced by the mitochondrial activity of cells, that measured at a wavelength of 540–630 nm and directly proportional to the number of living cells, the increase or decrease in the number of living cells is linearly related to the activity of cell mitochondria. MTT tetrazolium dye is revived in active (metabolically) cells. Mitochondrial dehydrogenases in living cells produce NADH and NADPH, leading to an insoluble purple precipitate called formazan. This precipitate can be dissolved by isopropanol or dimethyl sulfoxide (Lu et al., 2021). Dead cells, on the other hand, are unable to perform this conversion due to the inactivity of their mitochondria and therefore do not show a signal. In this method, dye formation is used as a marker for the presence of living cells. In recent years, MTT testing has been the most important measurement method to evaluate the toxicity and anti-cancer effects of metal nanoparticles (Lu et al., 2021).

In this study, infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cells were used to evaluate the anticancer effect of Cu NPs@CS/Alg nanocomposite on cell culture. For this purpose, each cell line was placed separately in T25 flasks with a complete culture medium (including DMEM (Dulbecco's Modified Eagle Medium, 10% complementary bovine fetal serum, and 1% penicillin–streptomycin solution) and at 37 °C in the incubator, cell culture was incubated with 5% CO₂.

After obtaining 80% cell density, the sample was exposed to 1% trypsin-EDTA solution and after 3 min of incubation at 37 °C in a cell culture incubator with 5% CO₂ and observation of cells removed from the bottom of the plate, the sample was centrifuged at 5000 rpm for 5 min and then the cell precipitate was decrypted by adding trypsin culture medium. Then, the cell suspensions after adding trypan blue dye were counted by neobar slide and cytotoxicity test was performed by MTT method. For this purpose, in each well of 98 cell culture plate, 10,000 KYSE-270, OE33, and ESO26 cells were introduced with 200 µl from the complete cell culture medium and to achieve the cell monolayer density, the plate was re-exposed to 5% CO₂ at 37 °C. After reaching 80% cell growth, the culture medium was removed and the cell surface was first washed with PBS buffer, again, in all wells, a complete two-concentration culture medium of 100 μl was introduced and 100 μl of a solution of Cu NPs@CS/Alg nanocomposite dissolved in PBS (mg/mL²) was introduced into well No. 1. After mixing the nanoparticles in the culture medium, 100 μl of it was removed and added to the second well. In the next step, 100 μl of the second well was removed after stirring the medium and added to well 3. This operation was performed up to well 11 and thus the amount of nanoparticles in each well was halved, respectively. Well No. 12 contained only one cell and complete culture medium of one concentration and remained as a control. The plate was again exposed to 5% CO₂ at 37 °C for 24 h and after 24 h the cytotoxicity was determined using tetrazolium dye. 10 μl of tetrazolium dye (5 mg/ml) was added to all wells, including the control, and the plate was exposed to 5% CO₂ at 37 °C for 2 h. The dye was then removed from the wells and 100 μl of DMSO (Dimethyl sulfoxide) was added to the wells, the plate was wrapped in aluminum foil and shaken thoroughly in a shaker for 20 min. Finally, cell survival was recorded in ELISA reader at 540 nm (Lu et al., 2021): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

Then, based on the absorption rate of each well and its comparison with the control, the inhibitory concentration of 50% (IC50) was obtained (Lu et al., 2021).

After collecting data, Minitab statistical software was used for statistical analysis. Evaluation of antioxidant results in a completely randomized design and comparison of means was Duncan post-hoc test with a maximum error of 5%. To measure the percentage of cell survival in factorial experiments with the original design of completely randomized blocks and compare the means, Duncan post-hoc test with a maximum error of 5% was used. The 50% cytotoxicity (IC50) and 50% free radical scavenging (IC50)) was estimated with ED50 plus software (INER, V: 1.0). Measurements were reported as mean ± standard deviation.

3.1 Structural characterization of synthesized Cu NPs@CS/Alg nanocomposite

A post-synthetic modification approach was followed in the strategically developed Cu NPs@CS/Alg nanocomposite. It was based on preparing a hydrogel by two biopolymers, chitosan and alginate, that then further applied for in situ synthesized Cu NPs following the green process. The final Cu NPs@CS/Alg nanocomposite was subsequently analyzed through several techniques, such as, FT-IR, SEM, TEM, EDX, XRD and ICP-OES.

In order to rationalize the stepwise synthesis of Cu NPs@CS/Alg nanocomposite, FT-IR spectra of CS, Alig, CS/Alg composite and Cu NPs@CS/Alg nanocomposite have been depicted in Fig. 1. The characteristic absorption bands of chitosan are represented in Fig. 1a as, 1028 cm⁻¹ (C—N stretching), 1382 cm⁻¹ (C—O stretching of CH₂-OH linkage), 1591 cm⁻¹ (N—H bending), 1648 cm⁻¹ (C⚌O stretching of amide linkage) and 3400–3500 cm⁻¹ (O—H and N—H stretching) (Fazaeli et al., 2010). In the IR spectrum of sodium alginate (Fig. 1b), the asymmetric and symmetric stretching frequencies of C⚌O from COONa appeared at 1618 and 1418 cm⁻¹. The aliphatic C—H stretching and O—H stretching vibrations appear at 2852 and 3512 cm⁻¹ respectively (Konda et al., 2014; Mazaahir et al., 2012; Kiasat et al., 2013). Fig. 1c, representing the FT-IR spectrum of CS/Alg composite is literally an amalgamation of both individual component peaks with the slight shifting of characteristic peaks, which is a clear sign of successful hydrogelation of the biomolecules. And finally in the spectrum of Cu NPs@CS/Alg nanocomposite, as seen in Fig. 1d, due to strong complexation of in situ synthesized Cu atoms with the surface functions like NH₂, OH, CHOH, CH₂OH, C⚌O etc, the corresponding IR absorption bands are observed to be shifted to higher or lower regions respectively.

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Fig. 1

FT-IR spectra of a) CS, b) Alig, c) CS/Alg and d) Cu NPs@CS/Alg.

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The innate microstructural features, morphology, size and shape of the as-synthesized Cu NPs@CS/Alg nanocomposite was resolved by SEM and TEM images. Fig. 2 shown the SEM image of the quasi-spherical Cu NPs@CS/Alg nanocomposite. Functional modifications can be imagined from the surface coating over the particles. To have an idea of the more detailed structure of the nanocomposite, TEM analysis was carried out (Fig. 3). The related outcomes also justify the SEM results. It reveals the perfectly spherical nanoparticles. The average diameter of the Cu NPs was around 10–20 nm. The particles are observed to be well dispersed without any sign of aggregation.

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Fig. 2

FE-SEM image of the Cu NPs@CS/Alg nanocomposite.

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Fig. 3

TEM images of the Cu NPs@CS/Alg nanocomposite.

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In order to have an idea of the chemical composition of the Cu NPs@CS/Alg nanocomposite, EDX analysis was carried out and the profile is shown in Fig. 4. The elements C, N, O and Na are attributed to CS/Alg composite. The presence of Cu element confirmed the successful fabrication of Cu NPs over CS/Alg composite. The results were further justified by SEM elemental mapping analysis (Fig. 5). The compositional map reveals the C, N and Cu species to exist with excellent dispersion throughout the matrix surface.

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Fig. 4

EDX spectrum of the Cu NPs@CS/Alg nanocomposite.

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Fig. 5

Elemental mapping of Cu NPs@CS/Alg nanocomposite.

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Finally, the phase structure and crystallinity of the prepared Cu NPs@CS/Alg nanocomposite was ascertained from XRD analysis (see Fig. 6). While the initial phase in the 2θ region up to 20° was attributed to CS/Alg composite and non-crystalline. Additionally, the several diffraction peaks appeared at the 2 Theta degree of 43.2°, 50.3° and 74.1° corresponds to the crystal planes (1 1 1), (2 0 0), and (2 2 0), respectively. All the peaks are authenticated standard data which that satisfies the highly pure crystalline Cu nanoparticles (JCPDS No. 04-0836).

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Fig. 6

XRD pattern of the Cu NPs@CS/Alg nanocomposite.

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3.2 Analysis of the antioxidant potentials of Cu NPs@CS/Alg nanocomposite

Oxidative stress is caused by an imbalance between the production of free radicals and metabolic reactions, which leads to damage to lipids, proteins and nucleic acids. These damages may be due to low levels of antioxidants or an excessive increase in the production of free radicals in the body (Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018). In humans, oxidative stress is associated with chronic diseases such as diabetes and cancer. Therefore, the production of synthetic and natural antioxidants is necessary to prevent oxidative stress and its destructive effects.

Antioxidants effectively and in various ways reduce the harmful effects of free radicals in the biological and food systems and cause detoxification (Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017). In this regard, green nanoparticles can be used (using plant substrates to prepare nanomaterials that are environmentally friendly and do not contain any harmful chemicals) that show antioxidant properties. At present, the use of non-toxic substances in synthesizing nanoparticles to prevent biological hazards, especially in medical and pharmaceutical applications is considered (Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018; Radini et al., 2018). Many researchers have focused on bioactive substances derived from plants or other sources such as bacteria, fungi and yeast for synthesizing nanoparticles. The green synthesis method is thought to increase the biocompatibility and performance of metal nanoparticles for biological applications due to removing harmful chemicals (Davis et al., 2008; Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018). During the bioproduction stages of nanoparticles, their extracellular production using plants or their extracts is more beneficial and their production can be adjusted in a controlled way based on size, distribution and shape for different purposes (Davis et al., 2008; Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018).

In the recent study, the scavenging capacity of Cu NPs@CS/Alg nanocomposite and BHT at different concentrations expressed as percentage inhibition has been indicated in Table 1 and Fig. 7. In the antioxidant test, the IC50 of Cu NPs@CS/Alg nanocomposite and BHT against DPPH free radicals were 334 and 193 µg/mL, respectively (Table 1).

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Fig. 7

The antioxidant properties of Cu NPs@CS/Alg nanocomposite (a) and BHT (b) against DPPH.

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3.3 Analysis of the anti-human breast cancer potentials of Cu NPs@CS/Alg nanocomposite

The unique physical and chemical properties of copper nanoparticles have attracted the attention of the scientific community due to their high plasmonic properties, heat transfer, chemical stability and antibacterial effects. Using copper is nothing new; it dates back to Hippocrates, who used it as an antibacterial to control wounds (Radini et al., 2018; Beyene et al., 2017; Chen and Schluesener, 2008). Today, copper nanoparticles are used in many commercial products, including soap, food, plastics, catheters, textiles, and bandages. However, their mechanism of action is still unknown. Many factors (shape, size, surface chemistry, morphology, density, charge, and purity) affect the biological activity of copper nanoparticles (Alexander, 2009; Bhattacharya, 2011; Jo et al., 2015). Another important feature of copper nanoparticles is their role in treating cancer. Copper nanoparticles are a promising tool as an anti-cancer agent in diagnosis and evaluation. They have many benefits with strong effects against different cancer cell lines. Their better penetration and ability to detect copper nanoparticles in the body make them a more effective tool in treating low-risk cancers compared to standard treatments (Bhattacharya, 2011; Jo et al., 2015; Rai et al., 2014; Riehemann et al., 2009). The unique properties of copper nanoparticles, such as their optical properties, easy synthesis and high surface-to-volume ratio, make them suitable for treating cancer. Copper nanoparticles can also be conjugated to various molecules, including DNA and RNA, to target different cells and antibodies or polymers. These important factors are important for increasing the half-life for circulating in vivo, which is very important in drug and gene delivery applications. In addition, copper nanoparticles are used as a cancer cell erosion tool because of their ability to convert radio frequency into heat (Jo et al., 2015; Rai et al., 2014; Riehemann et al., 2009; Huang et al., 2017; Conde et al., 2012).

Copper nanoparticles induce changes in cell morphology, decrease cell metabolic activity, increase oxidative stress leading to mitochondrial damage, and ultimately damage DNA by producing reactive oxygen species (ROS). Adsorption of copper nanoparticles occurs through endocytosis (Riehemann et al., 2009; Huang et al., 2017; Conde et al., 2012; Bhattacharyya et al., 2011). Examination of cancer morphology shows that synthesizing copper nanoparticles can significantly increase cell death. Now, if chitosan is combined with copper nanoparticles, it increases the rate of cell death. The mechanism of anti-cancer activity of copper nanoparticles is that copper nanoparticles induce apoptosis in cancer cells (Rai et al., 2014; Riehemann et al., 2009; Huang et al., 2017). Copper nanoparticles are capable of altering the regulation of more than 1,000 genes. Among these genes are metallothianonine, chaperones, and histones. Autophagy is one of the anti-cancer mechanisms of copper nanoparticles, which induces cell death (Huang et al., 2017; Conde et al., 2012; Bhattacharyya et al., 2011). Nanoparticle-induced autophagy is an important cell degradation process, and increased autophagy can also increase cell death. Recent studies show that copper nanoparticles can induce autophagy through the accumulation of autophagolysosomes in ovarian cancer cells. Therefore, autophagy can affect performance; At low levels it can increase cell survival and at high levels it can cause cell death. Another mechanism of copper nanoparticles is the increase in the level of internal ion concentration, which increases the production of ROS. On the other hand, uncontrolled production of ROS can lead to serious cellular damage, including DNA and mitochondrial damage, leading to programmed cell death (apoptosis) (Bhattacharyya et al., 2011; Sau et al., 2010; Sperling and Parak, 2010; Pelaz et al., 2015; Day et al., 2009). The cytotoxic activity of copper nanoparticles in breast cancer cells is through activation of caspase 3, p53, pErk1/2 and lack of Bcl2 expression. In particular, copper nanoparticles induce cell death through various processes including ROS production and increased lactate dehydrogenase secretion, induction of apoptosis, induction of autophagic genes, mitochondrial dysfunction and activation of caspase and DNA damage (Beyene et al., 2017; Chen and Schluesener, 2008; Alexander, 2009; Bhattacharya, 2011; Jo et al., 2015; Rai et al., 2014). Copper nanoparticles, like other biomaterials, can cause toxic effects in living organisms. The toxicity created by these particles depends on their properties and route of entry. The toxicity induced by copper nanoparticles is due to oxidative stress, which accumulates in the cytoplasm and cell nucleus, leading to the production of free radicals (Huang et al., 2017; Conde et al., 2012). Copper nanoparticles are initially separate particles that have the highest toxicity due to their high contact surface, but their toxicity decreases over time and joins together (Riehemann et al., 2009; Huang et al., 2017; Conde et al., 2012; Bhattacharyya et al., 2011; Sau et al., 2010).

In this investigation, the treated cells with different concentrations of the present Cu NPs@CS/Alg nanocomposite were assessed by MTT assay for 48 h about the cytotoxicity properties on normal (HUVEC) and breast malignancy cell lines i.e. infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) (Fig. 8).

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Fig. 8

The anti-human breast cancer properties (Cell viability (%)) of Cu NPs@CS/Alg nanocomposite (Concentrations of 0–1000 µg/mL) against normal (HUVEC: a) and human breast cancer (infiltrating lobular carcinoma of breast (UACC-3133 (b)), inflammatory carcinoma of the breast (UACC-732 (c)), and metastatic carcinoma (MDA-MB-453 (d))) cell lines.

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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 Cu NPs@CS/Alg nanocomposite (Table 2 and Fig. 8).

The viability of malignant breast cell lines reduced dose-dependently in the presence of Cu NPs@CS/Alg nanocomposite. The IC50 of Cu NPs@CS/Alg nanocomposite were 297, 386, and 359 µg/mL against KYSE-270, OE33, and ESO26 cell lines, respectively (Table 2).

It seems that the anti-human breast cancer effect of recent nanoparticles is due to their antioxidant effects. Because tumor progression is so closely linked to inflammation and oxidative stress, a compound with anti-inflammatory or antioxidant properties can be an anticarcinogenic agent (Namvar et al., 2014; Sankar et al., 2014).

Many nanoparticles have pharmacological and biochemical properties, including antioxidant and anti-inflammatory properties, which appear to be involved in anticarcinogenic and antimutagenic activities (Sankar et al., 2014; Katata-Seru et al., 2018). Today, nanoparticles synthesized by biological methods play a vital role in treating many diseases, including cancer (Katata-Seru et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018). Nanoparticles synthesized by biological methods are no longer the only ones in traditional medicine, in addition, they have been able to adopt an industrial line of natural products for treating various cancers. Various cell lines from cancers of the prostate, ovary, lung, liver, and pancreas have been treated with metallic nanoparticles (Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018).