Decorated CuO nanoparticles over chitosan-functionalized magnetic nanoparticles: Investigation of its anti-colon carcinoma and anti-gastric cancer effects

2.1 Preparation of aminopropyl silica-coated Fe₃O₄ NPs (Fe₃O₄-NH₂)

Magnetic nanoparticles were prepared via co-precipitation of Fe (III) and Fe (II) ions with a molar ratio of 2:1 in the presence of ammonium hydroxide. Generally, a mixture of FeCl₃·6H₂O (5.838 g, 0.0216 mol) and FeCl₂·4H₂O (2.147 g, 0.0108 mol) was dissolved in 100 mL deionized water at 85 ^°C under N₂ atmosphere and intense mechanical stirring (500 rpm). Afterwards, 10 mL of 25% NH₄OH was quickly injected into the reaction mixture in one section. The addition of the base to the Fe^2+/Fe³⁺ salt solution resulted in the formation of the black precipitate of MNPs instantly. The reaction continued for another 25 min and the mixture was cooled to room temperature. The magnetic nanoparticles as a dark solid were isolated from the solution by magnetic separation and washed several times by DI water.

1.0 gm Fe₃O₄ NPs were added in 50 mL toluene and sonicated ure for 20 min and stirred for 30 min. Then, 0.1 mL 3-aminopropyl trimethoxysilane (APTMS) was then injected to the resulting mixture using a microsyringe and stirred continued for 1 h prior to the injection of (0.1 mL) and finish the surface modification with aminopropyl group. The obtained hybrid material (Fe₃O₄-NH₂) was collected with an external magnet and washed with ethanol and deionized water for three times and dried at 60 °C.

2.2 Preparation of Fe₃O₄-NH₂@CS/CuO nanocomposite

In a typical process, Fe₃O₄-NH₂ particles (0.5 g) were dispersed in 50 mL deionized water. A 30 mL homogeneous mixture of chitosan (0.2 g CS in 50 mL 1% HOAc) was added to the mixture. The resulted mixture was stirred and subsequently a glutaraldehyde aqueous solution (5 wt%, 10 mL) was added and stirred again 60 min at 50 °C. The resulted Fe₃O₄-NH₂@CS was collected by an external magnet, washed with ethanol and dried at 60 °C. Finally, 0.5 gm of Fe₃O₄-NH₂@CS composite was dispersed in 100 mL H₂O and sonicated for 20 min. Subsequently, an aqueous solution of Cu(NO₃)₂ (0.2 mmol in 20 mL H₂O) was added to reaction mixture and stirred for 12 h at room temperature. The Fe₃O₄-NH₂@CS/Cu²⁺ precursors were then hydrothermally (100 °C for 12 hrs) reacted using basic solution (NaOH, 0.5 mol/L), in order to grow CuO nanocrystals in the crosslinked chitosan matrix. The Fe₃O₄-NH₂@CS/CuO nanocomposite was collected using an external magnet and washed with distilled water and dried at ambient temperature. The Cu load was 0.28 mmol/g which measured by ICP-OES.

2.3 Determination of anti-human gastro-duodenal cancers potentials of Fe₃O₄-NH₂@CS/CuO nanocomposite by MTT assay

The MTT assay is a procedure of colorimetric based on reducing and breaking of yellow tetrazolium crystals by the enzyme succinate dehydrogenase to form insoluble purple crystals. In this method, unlike other methods, the steps of washing and collecting cells, which often cause the loss of a number of cells and increase the work error, have been eliminated and all test steps from the beginning of cell culture to reading the results with a photometer are performed on a microplate, so the repeatability, accuracy and sensitivity of the test are high (Shaib et al., 2013; Raut et al., 2010). If the test is performed on cells attached to the plate, an appropriate number of cells (about 2000 cells) must first be cultured in each of the wells. Then we select the control and test wells and add the appropriate amount of mitogen or drug to the test wells and place the plate in the incubator for the required time so that the desired substance affects the cells (Raut et al., 2010; Lotfi and Veisi, 2019). At the end of the incubation time, discard the supernatant and add 200 μl of culture medium containing half an mg/ml of MTT solution to each well and put it again in a carbon dioxide incubator for 2–4 h at 37 °C. During incubation, MTT is regenerated by one of the enzymes of the mitochondrial respiratory cycle i.e., succinate dehydrogenase. The regeneration and breakage of this ring produce purple-blue crystals of formazan that are easily detectable under a microscope. At the end, the optical absorption of the resulting solution can be read at 570 nm and the cells number can be calculated using a standard curve. For each cell line, there is a linear relationship between the number of cells and the light absorption of the final solution. Therefore, to examine each cell type, a standard curve related to the same cell line must be drawn and used (Raut et al., 2010; Lotfi and Veisi, 2019; Lu et al., 2021).

In this experiment, following human normal and gastro-duodenal cancers cell lines were used to determine the anticancer and cytotoxicity activity of gastro-duodenal over the Cu(NO₃)₂, Fe₃O₄, and Fe₃O₄-NH₂@CS/CuO nanocomposite using MTT assay:

1. Human gastric cancer cell lines:

   1. KATO III

   2. AGS

   3. MKN45

2. Human colorectal carcinoma cell lines:

   1. Ramos.2G6.4C10

   2. HCT-8 [HRT-18]

   3. HCT 116

   4. HT-29

The cells were in RPMI1640 liquid culture medium containing 2 mM glutamine, 10% inactivated bovine fetal serum (FBS), solution of streptomycin (100 μg/ml) and penicillin (100 units per ml) at 37 °C and 5% CO₂ and 95% moisture were cultured and passaged so that the cells reached the desired number in terms of morphology and number (after 3–4 passages). After separating the cells from the flask surface, trypsin-EDTA (Gibco BRL, Scotland) counted and evaluated cell viability, and 3000 cells were cultured in 96-well wells without or with Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite. Morphological changes and general characteristics of the cell were assessed 24 h using an inverted microscope (Motic, AE31 model, China) (Lu et al., 2021).

The effect of cytotoxicity of the Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite was evaluated by MTT Methy Thiazol Tetrazolium colorimetric test. MTT (Sigma-Aldrich, USA) is a tetrazolium salt based on living cell mitochondrial dehydrogenase succinate enzyme, which converts the yellow solution of MTT to insoluble purple crystals of formazan that is soluble in dimethyl sulfoxide (Lu et al., 2021).

The cells began to grow in 75 cm square T-flasks in 15 mL of medium with 10⁶ cells initial number. After 72 h and covering the bottom of the flask with cell, the cell layer adhering to the bottom of the flask was separated enzymatically using trypsin-Versen and transferred to a sterile test tube was centrifuged at 1200 rpm for 10 min (Germany). Sigma, 3–30 k model). The cells were then resuspended in a new culture medium with a pasteurizer pipette and 100 μl of cell suspension without or with Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite was added to each well of the 96-well plate so that there were 3000 cells in each well. The plates were incubated for 24 h in an incubator (Germany, Memmert) to return the cells to normal from the stress of trypsinization. Then, suitable dilutions of the desired Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite were prepared and 100 μl of each dilution was added columnar to the plate wells and the cells were incubated for 37 h at 37 °C and 5% CO₂. After 72 h of adding the Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite to the cells, 20 μl of 5 mg/ml MTT solution was added to each well. The plates were incubated for 4 h after the required time, the culture medium containing MTT was carefully removed and 100 μl of DMSO was added to each well to dissolve the resulting formazan. After 10 min and shaking the plates using the plate shaker, the light absorption at 570 nm was read by ELISA Reader (Awareness Technology Inc, Stat Fax 2100, USA). Cells containing no Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite were considered as controlled light density and wells without cells and only RPMI1640 medium with bovine fetal serum were considered as blank (Lu et al., 2021). $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

Finally, linear regression was performed to obtain the IC50 level, which represents the concentration of the extract, which causes 50% inhibition of cancer cell growth (Lu et al., 2021):

3.1 Analysis of catalytic characterization data

The method of FT-IR spectroscopy is used for detecting unknown substances, determining the quality or uniformity of the sample, determining the amount of ingredients in a mixture, identify mixtures of organic and inorganic compounds provided they are both solid or liquid, thin layer analysis, analysis of adhesives, coatings and adhesive enhancers or binders, identification of polymers and polymer mixtures, analysis of solvents, cleaners and detergents unknown, percentage of decomposition or non-polymerization of polymers and paints due to heat, UV or other factors, determination of the degree of crystallization in polymers, and analysis of resins, composite materials and metal nanoparticles (Lotfi and Veisi, 2019; Lu et al., 2021; Rathore et al., 2014).

FT-IR spectra of the Fe₃O₄, Fe₃O₄-NH₂, CS, Fe₃O₄-NH₂@CS and Fe₃O₄-NH₂@CS/CuO are shown in Fig. 1 to confirm the successful synthesis of desired nanocomposite. Fig. 1a presents the representative peaks of bare Fe₃O₄ NPs, where the sharp bands at 582–637 cm⁻¹ correspond to Fe–O–Fe stretching vibrations. Fig. 1b shows a broad band at 3000–3500 cm⁻¹ attributing to OH and NH₂ stretching vibrations. A strong band at 550–650 cm⁻¹ indicates the presence of Fe-O bond. The broadband at 1100 cm⁻¹ can be assigned to Si-O-Si bond stretching vibration. The sp³ C-H stretching and NH₂ bending vibrations appear at around 2800 and 1550 cm⁻¹ respectively, indicating the aminopropyl attachment with the ferrite surface. FT-IR spectra of chitosan (Fig. 1c) shows a broad peak at 3429 cm⁻¹ corresponding to the overlapped stretching vibrations of NH₂ and OH groups. The alcoholic C–O stretching, asymmetric C–O–C (glycosidic) stretching, C–N stretching and N–H bending peaks are observed at 1380 cm⁻¹, 1156 cm⁻¹, 1027 cm⁻¹ and 1590 cm⁻¹ respectively. Fig. 1d shows the IR absorptions for Fe₃O₄-NH₂@CS material. It represents all the individual peaks from Fe₃O₄, silica layers, and chitosan indicating successful assembling. Only, the corresponding peaks are seen to be slightly moved to higher or lower regions. An additional peak is observed at 1635 cm⁻¹, attributed to the imine functionality (C⚌NH) formed during intermolecular cross-linking. Finally, in the analysis of Fe₃O₄-NH₂@CS/CuO nanocomposite we observed almost the same fashion as Fig. 1d except for very slight peak shifts of the imine, alcoholic C–O, C–N stretching vibrations to 1625, 1431, 1046 cm⁻¹ due to strong complexation of the corresponding functions to the Cu(II) NPs. The Fe-O bond vibration was also found moved to 564 cm⁻¹.

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Fig. 1

FT-IR spectra of a) Fe₃O₄, b) Fe₃O₄-NH₂, c) CS, d) Fe₃O₄-NH₂@CS and e) Fe₃O₄-NH₂@CS/CuO nanocomposite.

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Today, FE-SEM is used not only in materials science, chemistry and physics, but also in many fields such as medical and biological sciences. The high resolution of FE-SEM makes it one of the most-powerful and comprehensive tools for examining and analyzing a wide range of microstructure characteristics of samples at the nanometer to micrometer scale. Using the above technique, we can determine how the particles are placed from each other, their morphology and size (Shaib et al., 2013; Raut et al., 2010).

The morphological structure of the Fe₃O₄-NH₂@CS/CuO nanocomposite were studied by FE-SEM and TEM analysis. The globular shape nanoparticles can be detected from the FE-SEM image having a mean diameter of ∼20–30 nm (Fig. 2). The surface modification over Fe₃O₄ NPs by CS polymers can be adjudged from its appearances. There occurs a homogeneous growth of chitosan over it.

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Fig. 2

FESEM image of Fe₃O₄-NH₂@CS/CuO nanocomposite.

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EDX is a method that uses X-ray energy to analyze the structure and chemical composition of samples on a small scale. Using the EDX analysis method, qualitative and quantitative analysis can be performed on a wide range of metallurgical, biological, mineral and ceramic samples. Using the obtained information, it is possible to investigate the quantity and quality of specific phases and areas with homogeneous chemical composition. In other words, this method can be used for microanalysis (Shaib et al., 2013; Raut et al., 2010). In EDX analysis, by measuring the energy of the emitted rays, we can identify the type of element under study, which is a method of qualitative analysis. By measuring the intensity of X-rays, the concentration of sample elements can be determined, making it possible to quantitatively analyze unknown samples. The higher the observed ipak for the element in the graph, the higher the concentration of that element in the sample. Also, as the energy of the electron beam increases and the atomic weight of the elements decreases, more depth can be obtained from the sample information (Lotfi and Veisi, 2019; Lu et al., 2021).

EDX analysis of the Fe₃O₄-NH₂@CS/CuO nanocomposite exhibits Fe, Si, C, N, O and Cu species (Fig. 3). It confirmed the successful preparation of the desired nanocomposite. C and N atoms came from chitosan that coated on the surface of Fe₃O₄ nanoparticles.

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Fig. 3

EDX pattern of Fe₃O₄-NH₂@CS/CuO nanocomposite.

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The data obtained from EDX analysis were further rationalized through elemental mapping. X-ray scanning of a section of FESEM image reveals the uniform distribution of Fe, Si, C, N, O and Cu atoms over the nanocomposite surface (Fig. 4).

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Fig. 4

Elemental mapping of Fe₃O₄-NH₂@CS/CuO nanocomposite, Fe, Si, C, N and Cu respectively.

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One of the things that play a key role in the study of nanoparticles is determining their size. Transmission Electron Microscopy (TEM) is one of the most effective methods in determining particle size, which can provide us with useful quantitative and qualitative information. The TEM test is a method that allows direct imaging of particles up to the size of an atom, and this advantage of direct imaging must be considered when working with a microscope. Appropriate qualitative analysis of nanoparticles requires optimization of different imaging methods, magnification and manual or automatic analysis methods whose purpose is to optimize the image sharpness and contrast between the sample particles and the appropriate number of particles in each image, while minimizing damage to the sample. At lower image magnifications, it is possible to study the particle distribution, however, at high magnifications, a large number of particles are not observed and only information about the orientation of the plates and the structure is provided. In addition, at high magnification, high electron current causes instability and damage to the structure under study (Shaib et al., 2013; Raut et al., 2010; Lotfi and Veisi, 2019; Lu et al., 2021). The particle size distribution histograms for Fe₃O₄, Fe₃O₄-NH₂ and Fe₃O₄-NH₂@CS whose average size are approximately 15.8, 24.3 and 25.4 nm respectively (Fig. 5). This results confirmed the successful modification of materials for final composite.

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Fig. 5

The particle size distribution histograms for (a) Fe₃O₄, (b) Fe₃O₄-NH₂ and (c) Fe₃O₄-NH₂@CS.

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TEM study of the nanocomposite is presented in Fig. 5. The thin layer of chitosan bio-polymer surrounding the black colored Fe₃O₄ NPs can be detectable. The size of the final nanocomposite particles was in accordance with SEM study and is around ∼20–30 nm. The tiny black dots correspond to the Fe₃O₄ NPs. These data demonstrate the proposed architecture of the material. Moreover, Fig. 6 (inset) determines the particle size distribution histograms for Fe₃O₄-NH₂@CS/CuO nanocomposite whose average size are approximately 27.6 nm.

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Fig. 6

TEM images of Fe₃O₄-NH₂@CS/CuO nanocomposite and its particle size distribution histogram (inset).

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The saturation magnetization (Ms) value obtained from magnetic hysteresis loops of Fe₃O₄-NH₂@CS/CuO nanocomposite was 25.22 emu/g (Fig. 7). Still, the desired nanocomposite behaves like a superparamagnetic and can be easily isolated by an external magnet.

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Fig. 7

VSM analysis of Fe₃O₄-NH₂@CS/CuO nanocomposite.

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3.2 Anti-human gastro-duodenal cancers properties of Cu(NO₃)₂, Fe₃O₄, and Fe₃O₄-NH₂@CS/CuO nanocomposite

Despite many advances in disease control and treatment, cancer remains one of the global challenges to human health. The most common treatment for cancer is chemotherapy. An important point in the treatment of cancer with chemotherapy is the acquired resistance of the tumor to drugs, and this has created major problems for the treatment of cancer (Juul et al., 2018; Lauby-Secretan et al., 2016; Cunningham et al., 2010). Because the rate of mutation and genetic instability in cancer cells is very high and genetic changes occur rapidly in them, these cells become resistant to drugs. Therefore, further research on discovering new treatment strategies to overcome the drug resistance of cancers seems necessary (Shaib et al., 2013; Raut et al., 2010). Meanwhile, nanotechnology has created a promising field in cancer treatment. Recently, the anti-cancer and anti-angiogenic effects of metallic nanoparticles have been considered and the results have shown that metallic nanoparticles can be considered as a potential anti-cancer agent (Shaib et al., 2013; Raut et al., 2010; Lotfi and Veisi, 2019; Lu et al., 2021).

Many parameters such as surface functions nature and texture and size are important in the anticancer effects of metallic nanoparticles, of course the efficacy of the size is the main (Lotfi and Veisi, 2019). Many reports have been indicated whatever the nanoparticles size is low, their ability in poring and destroying the cancer cells is more. In detail, it has been reported the nanoparticles with the size lower than 50 nm have the best condition for anticancer effects (Lotfi and Veisi, 2019; Lu et al., 2021). As shown in the TEM images, the size of nanoparticles is less than 30 nm.

In the current experiment, the cytotoxicity of Cu(NO₃)₂, Fe₃O₄, and Fe₃O₄-NH₂@CS/CuO nanocomposite was explored by examining its interaction with human gastric cancer cell lines i.e. MKN45, AGS, and KATO III and human colorectal carcinoma cell lines.

The interactions being expressed as cell viability (%) was seen at several Cu(NO₃)₂, Fe₃O₄, and Fe₃O₄-NH₂@CS/CuO nanocomposite concentrations (0–1000 μg/mL) with the eight-cell lines which have been indicated in Figs. 8–10. In all the cases, the % cell viability gets decreased with raising Cu(NO₃)₂, Fe₃O₄, and Fe₃O₄-NH₂@CS/CuO nanocomposite samples concentrations. The IC50 of Fe₃O₄-NH₂@CS/CuO nanocomposite against MKN45, AGS, KATO III, HT-29, HCT 116, HCT-8 [HRT-18], and Ramos.2G6.4C10 cell lines were 517, 525, 544, 282, 214, 420, and 477 µg/mL, respectively (Tables 1 and 2). Thereby, the best anti-gastro-duodenal cancers findings of our Fe₃O₄-NH₂@CS/CuO nanocomposite was observed in the case of the HCT 116 cell line (Table 2).

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Fig. 8

The anti-gastric cancer properties of Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite against MKN45 (A), AGS (B), and KATO III (C) cell lines.

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Fig. 9

The anti-colorectal carcinoma properties of Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite against Ramos.2G6.4C10 (I), HCT-8 [HRT-18] (II), HCT 116 (III), and HT-29 (IV) cell lines.

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Fig. 10

The cytotoxicity effects of Fe₃O₄, Cu(NO₃)₂, and Fe₃O₄-NH₂@CS/CuO nanocomposite against Normal (HUVEC) cell line.

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Oxidation from reactive oxygen species can cause cell membrane disintegration, damage to membrane proteins, and DNA mutation that the result is the onset or exacerbation of many diseases (Shaib et al., 2013; Raut et al., 2010; Soni and Krishnamurthy, 2013; Katata-Seru et al., 2018; Sangami and Manu, 2017). Antioxidants with the property of removing free radicals play an important role in the prevention or treatment of oxidation-related diseases or free radicals. Extensive molecular cell research on cancer cells has developed a targeted approach to the biochemical prevention of cancers that the goal is to stop or return cells to their pre-cancerous state without any toxic doses through nutrients and drugs. Numerous studies have been performed on the use of natural compounds as anti-cancer agents in relation to appropriate antioxidant activity (Shaib et al., 2013; Raut et al., 2010; Lotfi and Veisi, 2019; Lu et al., 2021; Soni and Krishnamurthy, 2013; Katata-Seru et al., 2018; Sangami and Manu, 2017; Beheshtkhoo and Kouhbanani, 2018; Radini et al., 2018).

It seems the high anti-human gastro-duodenal cancers properties of Fe₃O₄-NH₂@CS/CuO nanocomposite are related to its antioxidant activities. Our successful efforts to utilize Fe₃O₄-NH₂@CS/CuO nanocomposite in gastro-duodenal cancers studies certainly shed light on future studies in this area.