Design and synthesis of nano Cu/chitosan-starch bio-composite for the treatment of human thyroid carcinoma

2.1 Preparation of CS-Starch composite the Cu NPs/CS-Starch bio-composite

Initially, the chitosan-starch composite (CS-Starch) was prepared by mixing chitosan (98% decetylated) and starch. In order of that, 0.5 g each of them were dissolved in100 mL of 1% (v/v) acetic acid solution by sonication for 30 min followed by stirring overnight at room temperature. The composite in the reaction mixture was used for the next step as such without any further processing.

2.2 Preparation of the Cu NPs/CS-Starch bio-composite

The CS-starch composite solution was made alkaline by addition of aq. NaOH solution (3 wt%) until pH 10. Subsequently, an aqueous solution of Cu(OAc)₂·2H₂O (40 mg in 10 ml) was added to it drop-wise over 10 min, under stirring at room temperature. Then the mixture was heated at 100 °C and stirred for another 5 h. Progress of the reaction could be monitored with the change in color of solution to grey due to the excitation of the surface plasmon resonance (SPR). The SPR band was a clear indication for the confirmation of Cu NP formation. The prepared Cu NPs/CS-Starch bio-composite were collected by centrifugation, washed several times with DI-water and dried at 40 °C. The Cu content was measured by ICP-OES analysis at 324.752 nm, to be 0.13 mmol/g.

2.3 Assessment of the antioxidant potential of Cu NPs/CS-Starch bio-composite by DPPH

Free radicals are usually unstable species having one or more unpaired electrons. These active species are biologically very harmful due to their high reactivity. They are most often formed when oxygen molecules in the body split into separate unstable atoms. This process can turn into a chain reaction. The excessive production of free radicals in the body causes cell damage and oxidative stress. Genetics and the internal environment of body influences the extent of free radical damage in individuals. These active molecules are produced as part of the body's natural biological processes. One of the most important free radicals is DPPH and widely used to determine the antioxidant potential of different natural compounds and NPs (Abbasi et al., 2021). In this process, DPPH radical scavenging capacity is used proportionally as the antioxidant activity of NPs. The basis of the action is the reduction of the alcoholic solution of DPPH in the presence of antioxidants. To achieve the IC50 of the samples, 11 different concentrations of NPs were prepared and the percentage of inhibition versus concentration was used to plot. In practice, 300 μL of 1 M DPPH was combined with 100 μL of diluted sample to a final volume of 2 ml using methanol. After half an hour in the dark, the absorbance was measured at 517 nm and the inhibitory percentage was obtained using the following formula: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}x100$$

In this formula, “Control A” shows the negative control of light absorption that lacks NPs, and “Sample A” expresses the amount of light absorption of different concentrations of NPs.

2.4 Measurement of cell toxicity of Cu NPs/CS-Starch bio-composite

Investigation of cell proliferation and survival is one of the most important and basic techniques in cell laboratories. This study requires accurate quantification of the number of living cells in the cell culture medium. Therefore, cell survival calculation methods are necessary to optimize cell culture conditions, evaluate cell growth factors, detect antibiotics and anticancer drugs, evaluate the toxic effects of environmental pollutants and study apoptosis. Several methods can be used for such purposes, but indirect methods using fluorescent dye (chromogenic) markers is very fast, particularly for large-scale samples. Among these, MTT (3-(4,5-dimethylthiazol-2-yl) −2,5-diphenyltetrazolium bromide) chromogenic method is the most widely used method for cell survival measurement. This is a colorimetric method to study cell proliferation and survival, introduced by Mossman in 1983. This is based on mitochondrial activity, which is stable in living cells and accordingly, a change in number of living cells is linearly related to mitochondrial activity. MTT tetrazolium dye is revived in active living cells. Mitochondrial dehydrogenases in living cells break down the tetrazolium ring and after the reaction with NADPH and NADH, leads to the formation a purple insoluble deposit, called formazan. This precipitate can be dissolved by dimethyl sulfoxide or isopropanol. On the other hand, dead cells do not have this ability and therefore do not reveal such a signal. Dye formation is used as a marker of living cells. The color intensity produced is measured at a 540 to 630 nm wavelength and is directly proportional to the living cells number. High safety and providing a colorimetric and non-radioactive system are significant advantages of this method. This kit is very easy to use, has high sensitivity and accuracy and can detect less than 950 cells. On the other hand, it has high efficiency for measuring cell proliferation, survival and mortality, and its implementation method does not require time-consuming washing steps and transfer from one plate to another. Examples studied in this method are adhesive or suspended cells and proliferating or non-proliferating cells. In our experiment, we used normal cell line (HUVEC) and Human thyroid cancer cell lines (TPC1, BCPAP, and FTC133) cell for investing the cytotoxicity and anti-human thyroid cancer effects of the Cu NPs/CS-Starch bio-composite using an MTT assay:

Because NPs are not soluble directly in 1640-RPMI medium and also the solvent of dimethyl sulfoxide nanoparticles (DMSO) itself has cytotoxic effects, to eliminate the effect of this substance on treated cells, its concentration in the final solution is considered < 1%. DMSO is not toxic to such lower concentration. For this purpose, 1000 µg of NP was dispersed in 100 μL DMSO after weighing. Then 1 ml of culture medium was added for better dissolution and finally the volume of solution was increased to 24 ml using culture medium. Then, successive dilutions of this stock were used in the range of 1–1000 μg/ml with 11 samples, being used for the cell lines. 100 μL of culture medium containing 10⁴ cells per plate 96 were placed. After 24 h of incubation, the NPs samples were added to the cell lines and incubated again for 24, 48, and 72 h, respectively. Subsequently, 20 μL of MTT plate with a concentration of 5 mg/ml was added to each cell and incubated in dark for another 4 h. After some time, the MTT medium was carefully removed and 200 μL of acidified isopropanol were added to each plate to remove the purple formazan. After 15 min of incubation at room temperature, the light absorption of each well was read using an ELISA at 570 nm against a reference wavelength of 690 nm. The outcomes were reported as cell survival and IC50 (concentration that inhibits cell growth up to 50%) based on the concentration curve (μg/ml). Notably, that the effect of each concentration of the nanoparticles on cell lines was investigated in five independent experiments. According to the values ​​of light absorption obtained by the ELISA reader, the percentage of growth inhibition related to each concentration was calculated using the following formula: $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{ControlA}.}x100$$

Finally, linear regression was done to gain IC50, which indicates the nanoparticles concentration, which causes 50% cancer cell growth inhibition. Using the curve, the line equation for cancer cells was obtained, respectively, then by replacing 50% inhibition in the equation, the IC50 value for cancer cells was obtained.

2.5 Statistical analysis

SPSS statistical software version 22 was used for data analysis and the outcomes were determined as the mean standard deviation of 5 replications. Data were analyzed using one-way analysis of variance and Duncan post hoc test and the significance level in the test was considered 0.05.

3.1 Analysis of catalytic characterization data

After the templated green synthesis of Cu NPs over chitosan-starch polyplex (Scheme 1), the next endeavor was to characterize the as synthesized material in order to study its physicochemical features and structural morphology. We investigated the FT-IR, SEM, TEM, EDX, XRD, UV–Vis, TGA and ICP-OES analyses over the Cu NPs/CS-Starch bio-nanocomposite.

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Scheme 1 Synthetic scheme of Cu NPs/CS-Starch bio-composite.

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Fig. 1 describes the ensemble of FT-IR spectrum of Cu NP/CS-starch nanocomposite (c) and its two precursor chitosan and starch (a and b) to justify construction of final material. The characteristic peaks of chitosan are demonstrated in Fig. 1a, where the bond vibrations of C–N (C-NH₂), C-O-C stretching (glycosidic), C–O stretching (CHOH and CH₂-OH), N-H bending (NH₂), C = O (CONH₂) stretching and C-H stretching are observed at 1032 cm⁻¹, 1383–1456 cm⁻¹, 1601 cm⁻¹, 1649 cm⁻¹ and ∼ 2900 cm⁻¹ respectively. The broad band appeared at 3400–3500 cm⁻¹ corresponds to the combined N-H and O-H stretching vibrations. By structure, chitosan and starch are very much alike, only starch lacks the amino linkages. Hence, the bonding vibrations of starch resemble closely to that of chitosan, being presented in Fig. 1b. As predicted, the peaks due to C-N and N-H bond vibrations are not observed in the FT-IR profile of starch. Finally, the FT-IR spectrum of Cu NP/CS-starch comprises all of its precursor component bond vibrations from chitosan and starch, signifying the successful incorporation of them in the composite (Fig. 1c). Nevertheless, there occurs a slight relocation of the peaks in Fig. 1c as compared to 1a and 1b, which is anticipated due to strong complexation of Cu NPs with the surface polyplex.

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Fig. 1 FT-IR spectra of a) CS, b) Starch and c) Cu NPs/CS-Starch bio-nanocomposite.

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UV–Vis spectroscopy is a potential measure for the identification of Cu NPs. The spectrum recorded intervals of the reaction are presented in Fig. 2. After 5 h, the reaction was ended with the formation of Cu NPs which was confirmed by the characteristic surface plasmon absorbance (sky blue line) at 589 nm (Shi et al., 2021).

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Fig. 2 UV–Vis spectrum of Cu NP/CS-Starch.

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Surface morphology, shape, size and the texture of Cu NP/CS-Starch nanocomposite were evaluated by FE-SEM study, as presented in Fig. 3. As it's a hydrogel based nanocomposite (CS-Starch), the appearance of FE-SEM image is like a sheet. The unlikely texture might be due to manual sample preparations. However, Cu NPs could not be separately detected in the image.

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Fig. 3 FE-SEM image of Cu NPs/CS-Starch bio-nanocomposite.

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An EDX analyzer attached with the FE-SEM equipment was used to have knowledge of the molecular composition of Cu NPs/CS-Starch bio-nanocomposite. As depicted in Fig. 4, the material contains Cu as the metallic and C, N and O as non-metallic components. The peak appeared at 0.9 eV, represents the Cu species, being evidenced from earlier reports. The strong peak of Au appeared by default due to pre-treatment of the material under Au vapor. The non-metals correspond to the attachment of chitosan and starch with Cu.

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Fig. 4 EDX spectrum of Cu NPs/CS-Starch nanocompsite.

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A comprehensive fundamental structure analysis and surface morphological study of the Cu NPs/CS-Starch nanocomposite was carried out by TEM investigations (Fig. 5). Prior to the analysis, the sample was uniformly dispersed in acetone by sonication and then the dispersed solution was dropped over a carbon coated Cu grid, aerially dried and finally analyzed under microscope. The tiny black dots represent the surface modified Cu NPs. They are perfectly round shaped and of equal size. The mean size of the particles is assessed to be 5–7 nm. However, the surface shell of chitosan-starch composite over the Cu NPs is hard to detect from the TEM image.

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Fig. 5 TEM image of Cu NPs/CS-Starch bio-nanocomposite.

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Crystallinity and phase structure of the Cu NPs/CS-Starch bio-nanocomposite was determined by XRD study and the corresponding profile has been depicted in Fig. 6. It displays a single phase profile signifying a sole entity. The broad non-crystalline regions up to 2θ = 25° are ascribed to the CS-Starch combined polyplex. The encapsulated Cu NP seems to be poorly crystalline. Bragg's diffraction peaks of Cu NPs are observed at 2θ = 29.76°, 36.46°, 42.38°, 61.32°, 73.81°, and 77.18° which are attributed to the reflections on (1 1 0), (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes related to cubic Cu NPs (JCPDS Card No. 05–0667) (Kuo and Huang, 2008).

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Fig. 6 XRD pattern of Cu NPs/CS-Starch bio-nanocomposite.

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The stability of the synthesized Cu NPs/CS-Starch bio-nanocomposite and also percent of organic functional groups on the composite were studied using TGA analysis. The composite TGA curve (Fig. 7) shows a primary weight loss of 7.6% up to 185 °C which is because of physically adsorbed solvent and surface hydroxyl groups on the support. Thermal degradation of the composite happened following 200 °C which shows the hh thermo-stability of composite. The second and third weight loss of 33% over the range of 200–600 °C was due to degrading the organic remains.

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Fig. 7 TGA curve of Cu NPs/CS-Starch bio-nanocomposite.

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3.2 Antioxidant properties of Cu NPs/CS-Starch bio-nanocomposite

Oxidation is the electron transfer from an atom and this is the aerobic life and metabolism part of living organisms. Oxygen is the receptor for electrons in the electron transport system, which yields energy from ATP (Adenosine triphosphate) in the body. Under certain conditions, oxygen may become a single electron species and generate free radicals, which is also called as reactive oxygen species (ROS). Oxidative reaction of proteins, DNA, and other biological macromolecules is one of the internal causes of degenerative diseases such as aging, cardiovascular disease, cancer, immune system deficiency, cataracts, and abnormal brain function. High-energy, mutagenic, single electron oxygen, can be produced by lipid peroxidation by the transmission of light energy from light (Abbasi et al., 2021). Some free radicals have positive roles such as regulating cell growth, phagocytosis, energy production, intracellular signals, or the important biological compounds synthesis. Antioxidants produced in the body fight the free radicals following two systems, such as, enzymatic defense and non-enzymatic defense. Superoxide dismutase, catalase and glutathione peroxidase metabolize lipid peroxide, hydrogen peroxide, and superoxide and prevent the production of toxic hydroxyl radicals (Zangeneh et al., 2019). In non-enzymatic defense, there are two classes of fat-soluble antioxidants (such as carotenoids and vitamin E) and water-soluble (glutathione and vitamin C) that trap free radicals. These two systems help neutralize oxidants. However, oxidants can escape from antioxidants and damage tissues. In this case, the activated antioxidant repair system (which is the enzymes lipase, protease, transferase and DNA repair enzymes), counteract the oxidant effects. However, due to deficiencies in the production of antioxidants in the body or due to physiopathological factors and situations (such as smoking, air pollution, UV radiation, diets containing high unsaturated fatty acids, inflammation, ischemia, bleeding, etc) that ROS are produced in large quantities at the wrong place and time and hence oral antioxidants are needed to counteract the oxidative damage cumulative effects (Zangeneh et al., 2019; Zangeneh et al., 2019; Jalalvand et al., 2019).

In the present study, antioxidant effects of the Cu NPs/CS-Starch bio-nanocomposite, evaluated by DPPH assay, revealed concentration-dependent effects i.e., an increase in the concentration of the Cu NPs/CS-Starch bio-nanocomposite leads to an increase in antioxidant activities. In the concentrations studies, the best result was observed at high concentrations or 1000 µg/mL (Fig. 8). Comparative analysis of the individual antioxidant assays showed significant variations in the exertion of radical scavenging effects. In contrast, standard (butylated hydroxytoluene) demonstrated lower antioxidant effects compared to the Cu NPs/CS-Starch bio-nanocomposite.

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Fig. 8 The antioxidant properties of Cu NPs/CS-Starch bio-composite (A) and BHT (B) against DPPH. The numbers indicate the percent of free radical (DPPH) inhibition at the concentrations of 0–1000 μg/mL of Cu NPs/CS-Starch bio-composite (A) and BHT (B).

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The IC50 of Cu NPs/CS-Starch bio-nanocomposite and BHT were 207 and 143 µg/mL, respectively (Table 1).

3.3 Cytotoxicity and anti-human thyroid cancer potentials of Cu NPs/CS-Starch bio-composite

Recently, the anti-angiogenic and anti-cancer properties of metallic nanoparticles have been determined and the results revealed that they can be used as a unique anti-cancer supplement. But the organic solvents used to produce these nanoparticles are toxic and can have devastating environmental effects. Hence, there is a huge demand of healthy and sustainable methods for synthesizing metallic nanoparticles. Recently, green chemistry studies have opened up an arena for the environmental friendly pathway towards the synthesis of nanoparticles (Abbasi et al., 2021). In recent years, metal nanoparticles have been synthesized extracellularly using various plant and microbial extracts. While exploring the applications of these 'green' products, several research groups have been working to investigate their antibacterial, antioxidant, anticancer and anti-tumor properties (Zangeneh et al., 2019; Zangeneh et al., 2019; Jalalvand et al., 2019). The anti-inflammatory and antimicrobial effects of biogenic metallic nanoparticles are well known (Zangeneh et al., 2019). These materials also affect a range of molecular targets and signaling pathways, such as NF-JB, AKT/mTOR, and HIF-1A, and as a result, it plays a significant role in the inhibition of cancer cell proliferation, metastasis, angiogenesis and also inducing apoptosis. There are earlier reports of biogenic metallic nanoparticles causing deformity and perforation of cancer cells, resulting in their death (Zangeneh et al., 2019; Zangeneh et al., 2019).

In our studies of cytotoxicity measurement, the normal (HUVEC) and human thyroid cancer cell lines i.e., TPC1, BCPAP, and FTC133 were treated with several concentrations of Cu NPs/CS-Starch bio-composite followed by MTT test for 48 h (Fig. 9 and Table 2). The absorbance rate was determined at 570 nm, which indicated extraordinary viability on normal cell line (HUVEC) even up to 1000 μg/mL for Cu NPs/CS-Starch bio-composite. Interestingly, viability of the corresponding thyroid cell lines reduced dose-dependently over the Cu NPs/CS-Starch bio-composite. The IC50 of Cu NPs/CS-Starch bio-composite against TPC1, BCPAP, and FTC133 cell lines were 269, 115 and 148 µg/mL, respectively. The best result of anti-human thyroid cancer property of Cu NPs/CS-Starch bio-composite against the above cell lines was observed in the BCPAP cell line.

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Fig. 9 The anti-human thyroid carcinoma properties (Cell viability (%)) of Cu NPs/CS-Starch bio-composite (Concentrations of 0–1000 µg/mL) against normal (HUVEC: A) and human thyroid carcinoma (TPC1 (B), BCPAP (C), and FTC133 (D)) cell lines. The numbers indicate the percent of cell viability at the concentrations of 0–1000 μg/mL of Cu NPs/CS-Starch bio-composite against several human thyroid carcinoma cell lines. sa.

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