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Back to Journal »Drug Design, Development and Treatment» Volume 15

Ethyl cellulose coated with silver nanoparticles inhibits tumor necrosis factor-α in breast cancer cells

Author: Abdellatif AAH, Alsharidah M, Al Rugaie O, Tawfeek HM, Tolba NS

Published on May 13, 2021, the 2021 volume: 15 pages 2035-2046

DOI https://doi.org/10.2147/DDDT.S310760

Single anonymous peer review

Editor approved for publication: Dr. Deng Tuo

Ahmed AH Abdellatif,1,2 Mansour Alsharidah,3 Osamah Al Rugaie,4 Hesham M Tawfeek,5 Nahla Sameh Tolba6 1 Department of Pharmacy, School of Pharmacy, Qasim University, Buraydah, 51452, Saudi Arabia; 2 Al-Azhar University School of Pharmacy Department of Pharmacy and Industrial Pharmacy, Assiut, 71524, Egypt; 3 Department of Physiology, Faculty of Medicine, Qasim University, Buraydah, 51452, Saudi Arabia; 4 Department of Basic Medicine, Faculty of Medicine and Medical Sciences, Qasim University, Uniza, Alka Sim, 51911, Saudi Arabia; 5Department of Industrial Pharmacy, School of Pharmacy, Assiut University, Assiut, 71526, Egypt; 6Department of Pharmacy, School of Pharmacy, Sadat City University, Sadat, Egypt Newsletter: Ahmed AH Abdellatif School of Pharmacy, Qasim University , Buraydah, 51452, Saudi Arabia email [email protected] Introduction: Cancer is one of the leading causes of death worldwide. In many cases, cancer is associated with increased expression of an important cytokine called tumor necrosis factor-α (TNF-α). In particular, breast cancer is associated with increased tumor cell proliferation, high incidence of malignant tumors, more metastases, and general poor prognosis of patients. This study aimed to evaluate the effect of silver nanoparticles reduced with ethyl cellulose polymer (AgNPs-EC) on the expression of TNF-α in MCF-7 human breast cancer cells. Method: Green synthetic reduction method was used to produce AgNPs-EC, and its formation was proved by UV-VIS spectroscopy. In addition, AgNPs-EC are characterized by their size, charge, morphology, Ag ion release and stability. MCF-7 cells were treated with AgNPs-EC. Then, the expression of TNF-α gene was measured in real time by PCR, and protein expression was studied using ELISA. Results: AgNPs-EC is spherical, with an average size of 150±5.1 nm, and a zeta potential of -41.4±0.98 mV. AgNPs-EC has an inhibitory effect on cytokine mRNA and protein expression levels, which indicates that they can be used safely in anti-cancer. The cytotoxicity of AgNPs-EC was also found to be non-toxic to MCF-7. Conclusion: Our data confirm that AgNPs-EC is a new type of TNF-α inhibitor. These results are expected to develop new treatment methods for the use of safe materials to treat cancer in the future. Keywords: silver nanoparticles, tumor necrosis factor-α, ethyl cellulose, MCF-7 cells

Cancer is a very threatening disease and it spreads all over the world. It requires rapid and effective treatment to reduce its severity and ultimately improve the patient's prognosis. There is no doubt that nanotechnology has a major impact on finding many outstanding ways to overcome this disease and improving the shortcomings of conventional chemotherapeutics. 1 Tumor necrosis factor-α (TNF-α) is part of the TNF superfamily. It is the main cytokine used in cancer biotherapy. In many studies, TNF-α is used systematically to control and treat solid tumors, but it has shown serious toxicity, such as severe hypotension and organ failure. A lot of evidence shows that the pathophysiological concentration of endogenous TNF-α can stimulate the occurrence and growth of tumors. 2 Generally, in the treatment of breast cancer, TNF-α has a dual function, either as a target or as a drug. TNF-α has tumor-promoting and anti-tumor effects, depending on the cell environment, tumor cell characteristics and the origin of TNF-α. TNF-α is physiologically produced by tumor cells and stromal cells in the tumor microenvironment. 1

TNF-α is responsible for stimulating the proliferation, morphogenesis and differentiation of natural breast tissue. TNFR1 is a typical epithelial cell proliferation receptor, while TNFR2 changes the accumulation of casein. 3 It is reported that serum TNF-α in patients diagnosed with late-onset breast cancer is elevated, and it is related to the increase in the number and size of metastatic sites. 4 The increase in TNF-α levels may be related to the activation of the nuclear factor kappa-light chain enhancer of activated B cells (NF-#x1D6CB;B), which plays an important role in carcinogenesis and inflammation. 5 According to a study conducted by Wenliang et al.6, elevated TNF-α expression indicates poor results with sorafenib after surgery in patients with hepatocellular carcinoma (HCC). However, in vitro experiments have shown that TNF-α promotes the resistance of HCC cells to sorafenib by stimulating epithelial-mesenchymal transition (EMT). Several recent studies have shown that the pre-metastasis effect of TNF-α and its involvement in the EMT process that is essential for tumor cell migration and metastasis. Long-term exposure of breast cancer cell lines to TNF-α induces the up-regulation of Twist1 by activating IKKβ and NF-#x1D6CB;B transcription repressor. It stimulates EMT and stem cell cancer. 3 Another study mediated by Wu et al. 7 investigated whether TNF-α can improve the effects of chemotherapy and radiology on breast cancer cells. The study confirmed that TNF-α has radiotherapy and chemotherapy sensitization effects on breast cancer cells. TNF-α causes cells to leave the G0/G1 phase and enter the proliferation phase. Therefore, after irradiation, TNF-α causes more DNA damage and increases cell cycle arrest in G2/M and S phases induced by docetaxel and cisplatin, respectively. Fehaid and Taniguchi studied the influence of silver nanoparticles (AgNPs) as a molecular mechanism on the response of lung epithelial cells to TNF-α. Studies have shown that TNF-α can induce an increase in the uptake of AgNPs by cells, and a low AgNP concentration can enhance the protective effect of TNF-α on cell apoptosis. 8 Among them, metal nanoparticles have shown promise in fighting cancer and improving patient health. 9-11 AgNPs have proven to have excellent properties suitable for various applications, especially in biomedical applications. 12,13 AgNPs can treat cancer by changing cell morphology, reducing metabolic activity and cell viability, increasing oxidative stress, and increasing the production of reactive oxygen species (ROS) for DNA damage). 14 Asharani et al. Cellular and molecular mechanisms of the effects of different types of cancer cell lines, normal human lung cells, IMR-90 and human brain cancer cells, U251.15 AgNPs can adsorb the surface of cytoplasmic proteins, it affects the function of certain intracellular variables and regulates gene expression And pro-inflammatory cytokinesis. Another possible mechanism is that autophagy induced by AgNPs induces cell death, which is a key cell degradation process that leads to cell death when the level of autophagy increases. 16 Researchers investigated the development of AgNPs for effective cancer treatment, investigation, and diagnosis. Different drugs, polymers and nanomaterials can be conjugated and/or decorated with AgNPs to improve their selectivity and efficiency for other cancer cells. 17,18 Various materials can be used to reduce silver nitrate and stabilize the formed AgNPs, such as trisodium citrate and sodium borohydride. In addition, polymeric materials can also develop and stabilize AgNPs, such as PEG, chitosan and cellulose polymers. 19-21 These materials stabilize the formed AgNPs and enhance and improve the characteristic activity of the produced AgNPs. Muhammad et al. have demonstrated the effective anticancer activity of PEG-capped methotrexate AgNPs on MCF-7 cell lines, and reduced toxicity compared with methotrexate alone. twenty two

Azizi et al. have demonstrated the anticancer effect of albumin-coated AgNPs on MDA-MB 231 human breast cancer cells. 23 The authors revealed that AgNP-coated albumin is more cytotoxic to cancer cells than normal cells and cell death based on apoptosis, and that mouse gland tumors are reduced. Priya et al. explored the anticancer effect of biological AgNPs/chitosan on human hepatocellular carcinoma HepG2 cells. 24 Chitosan-coated AgNPs enhanced the apoptotic activity in vitro and had cytotoxic effects on the HepG2 cell line. Fahrenholtz et al. discussed the effects of using PVP-capped AgNPs as a single drug or in combination with cisplatin in the treatment of ovarian cancer. 25 The author described that AgNPs-PVP is extremely cytotoxic to A2780 and SKOV3 cell lines, but is not very sensitive to OVCAR3. However, the combination with cisplatin showed a synergistic effect on A2780 and OVCAR3 cells.

So far, according to our search in the literature, the effect of EC-coated AgNPs on cancer cells has not been studied earlier. This work aims to study AgNPs-EC targeting the MCF-7 breast cancer cell line. AgNPs are prepared by reducing silver nitrate using EC as a reducing agent and stabilizer. UV-VIS spectrophotometry was used to characterize the prepared AgNPs-EC to confirm its formation. In addition, the size, charge and morphology of AgNP were also studied. The in vitro release and physical stability of silver cations from AgNPs were also evaluated for 3 months. The MTT assay was used to study the cytotoxicity of AgNP-EC to MCF-7 cells after 24 hours of treatment. In addition, real-time PCR and ELISA were used to evaluate the effects of AgNP-EC MCF-7 proliferation, gene expression, and TNF-α production.

Sodium chloride, sodium hydroxide, nitric acid, hydrochloric acid, sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Merck (Darmstadt, Germany). 2,2-Diphenyl-1-picrylhydrazine (DPPH), MTT assay reagent and human TNF-α antibody were purchased from Sigma Aldrich (Steinheim, Germany). Cytokine-specific enzyme-linked immunosorbent assay was purchased from Santa Cruz Biotechnology Inc. (Bergheimer, Heidelberg, Germany). MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All chemicals are of analytical grade. All glassware was washed with distilled water and dried in an oven at 40°C.

The method previously reported by Abdellatif et al. was used to prepare AgNPs with EC stabilization. 26-28 Simply put, the EC standard 1% solution is prepared in distilled water. A 1 mM AgNO3 aqueous solution is also prepared and its pH is adjusted (8.0) and 1 mL/mol NaOH is used to determine the ionic strength. Heat the prepared AgNO3 solution on the hot plate to boiling, and continue stirring after adding 2 mL of the prepared EC solution. After about 20-30 minutes, the color of the solution changed to dark brown, indicating the formation of AgNPs-EC. Cool the solution to laboratory temperature. Finally, the solution was filtered by centrifugation at 3000 rpm for 5 minutes to extract concrete aggregate NP. The remaining filtrate was kept at 4.0 °C for further NP analysis. 29

To check the formation of AgNPs, we used UV-VIS scanning. Compared with the blank AgNO3 solution, a dual beam spectrophotometer (λ 25 UV-Vis spectrophotometer, PerkinElmer, Singapore) is used in the 300-600 wavelength range.

As mentioned earlier, the size and charge of the generated AgNPs-EC were calculated using Malvern Zetasizer Nano ZS (Malvern Instruments GmbH, Herrenberg, Germany). 26 The data is expressed as the average of three different measured values ​​of the same NP batch.

Transmission electron microscope is used to study the morphology of AgNP-EC (JEM-1230, Joel, Tokyo, Japan). AgNPs were fixed on a carbon-coated 300-mesh copper net, and then dried overnight. Use 10-100 K magnification and 100 kV acceleration voltage to observe AgNPs-EC. 30,31 Scanning electron microscope (SEM) (sputter coater, JOEL JFC-1300) is also used to study AgNPs-EC. The AgNP-EC solution was spotted on a 300-mesh copper mesh coated with carbon and then left on the carbon substrate. With the help of filter paper, remove the excess solution, and then rinse the copper grid twice with distilled water for 2-4 seconds each time. Then apply a 2% uranyl acetate aqueous solution and remove the excess solution; finally, the sample is allowed to dry at room temperature. 30 A 10-100 K magnification and 100 kV acceleration voltage are used to display the sample.

As our research team mentioned earlier, silver ions are released from the prepared AgNPs-EC. 26,32 The in vitro release was carried out in approximately 100 mL of double distilled water for 48 hours. Inductively coupled plasma spectroscopy (ICP-OES, iCAP 6000, Thermo Scientific, USA) and emission detector were used to calculate the amount of silver cations released at different time points against the blank silver metal standard solution. The data is expressed as the average of three additional readings.

The physical stability of the AgNP-EC dispersion is carried out under two different conditions, that is, the room temperature is 4.0±0.5 °C. 33 Prepare fresh samples and place them for 3 months under the conditions described above. Then, analyze the size, charge, and shape of the sample.

The research was conducted on a human breast tumor cell line (Michigan Cancer Foundation-7 (MCF-7)). Cells and 10% fetal bovine serum were cultured in RPMI-1640 solution containing antibiotics (100 u/mL penicillin and 100 μg/mL streptomycin) and seeded in 96-well plates at a density of 1×104 cells/well Medium, and at 37 °C, 5% CO2, 24 hours. 34 The MCF-7 cell line was incubated with AgNPs-EC at different concentrations (0.1-200 μM). The effect of AgNPs-EC on the viability of MCF-7 cell line was determined in vitro. CytoTox-Glo™ Cytotoxicity Detection Kit (Promega, Madison, Wisconsin, USA) was used to study cytotoxicity. 35 Then, different concentrations of AgNPs-EC (0.1–20 µM) were incubated with the MCF-7 cell line for 2 hours. The method of Abdellatif et al. is applicable to all formulations. 36 Remove the medium, and place the cells at a concentration of 150 μL/well of 10% trichloroacetic acid at 4°C for 1 hour, and then rinse with PBS pH 7.4 3 times. Stain the wells in the dark at 37 °C with 70 μL/well 0.4% SRB for 12 minutes. Remove the unbound dye, wash the cells with 1% acetic acid, and then air dry for 24 hours. In a shaker, dissolve the dye in 50 μL/well of 10 mM Tris base (pH 7.4) at 1600 rpm for 5 minutes. Calculate the optical density (OD) of each well at 570 nm (EXL 800 USA) using an ELISA microplate reader spectroscopically. According to the exponential viability curve to the standard concentration, the inhibitory concentration is estimated to be 50% (IC50). The viability of each compound is selected by the variable Sigmaplot software (Systat Software Inc), which is (sample A570/untreated A570)×100 and the IC50 amount of each compound (the concentration required to inhibit cell viability by 50%). In order to predict the cell viability and growth effect of the test compound, data were collected and analyzed.

Quantitative real-time PCR is used to measure mRNA expression, and GAPDH is used as an endogenous control, as defined in previous studies. 34 miRNA isolation kit is used to test total RNA (Thermo Fisher Scientific, Waltham, MA, USA). Superscript First-Strand cDNA Synthesis Kit (Sigma Aldrich, Schnelldorf, Germany) is used for reverse transcription of total RNA (0.5-2 μg). TaqMan gene expression analysis quantifies TNF-α mRNA expression (Thermo Fisher Scientific). Use the first-stage real-time PCR method (Applied Biosystems) for real-time PCR amplification and data capture, the initial stage is 95 °C, 10 minutes, the second step, 95 °C/17 seconds, 60 °C 60 seconds (40 cycles ). The target mRNA level is normalized to GAPDH and compared with the control (untreated sample). Analyze the obtained data using the ΔΔCT method. 37

The MCF-7 cell line was incubated with different doses of AgNPs-EC (0.1-20 μM) for 2 hours. According to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA), TNF-α specific ELISA was used to measure TNF-α in the culture medium. The ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) was used in accordance with the manufacturer's instructions. They use an automatic microplate reader (Anthos Zenyth 3100 Multimode Detectors, Salzburg, Austria). As described in the literature, read the microtiter plate at 460 nm. 34

A previous study conducted by our group showed the applicability and practicality of cellulosic polymers in the effective preparation of AgNP. EC shows the best in terms of higher antioxidant and antibacterial activity as well as excellent physical stability, size and charge. This is why EC was chosen in this study. 26 Size analysis and zeta potential measurement show that the size of the AgNPs-EC generated is 150±5.1 nm (Figure 1A) and the charge is -41.4±0.98 mV (Figure 1B). As discussed in different studies before, zeta potential The high value of reveals the stability of the dispersion produced. 26 The morphological observation of the produced particles was done using SEM (Figure 1C) and TEM (Figure 1D). Both of these techniques revealed the non-aggregated nature and spherical shape of the AgNPs-EC produced. In addition, TEM showed that the NP size was 39.3±5.8 nm, which confirmed the DLS and SEM results. Compared with DLS, the smaller size observed by TEM is a common phenomenon observed by other researchers. 30,38,39 Figure 1 AgNP reduced with ethyl cellulose. (A) The size distribution of the average diameter determined by DLS. (B) Apparent zeta potential determined by DLS. (C) SEM inspection. (D) Observed by transmission electron microscope.

Figure 1 AgNP reduced with ethyl cellulose. (A) The size distribution of the average diameter determined by DLS. (B) Apparent zeta potential determined by DLS. (C) SEM inspection. (D) Observed by transmission electron microscope.

The prepared AgNPs-EC is prepared by reduction of ethyl cellulose using silver nitrate. Ethyl cellulose is used as a reducing agent and stabilizing compound for effective AgNP preparation with almost no aggregation, as previously studied by different researchers. . 26,40 This cellulose derivative containing reducing groups and hydroxyl groups can provide silver nitrate with reducing properties to produce silver nanoparticles. The negatively charged EC helps to attract the positively charged silver cations to the polymer chain, which is then reduced with exciting reducing groups. Successful synthesis involves two important steps; as mentioned earlier, atom formation and atom polymerization. 41 The negatively charged EC helps to attract the positively charged silver cations to the polymer chain, which is then reduced with exciting reducing groups. Successful synthesis involves two important steps; as mentioned earlier, atom formation and atom polymerization. 42

Due to the surface plasmon resonance (SPR) effect of AgNPs and the successful reduction of silver ions to Ag in the water phase, UV-VIS spectrophotometric analysis showed a high absorption peak at a wavelength of about 391±0.16 nm 43, 44 (Figure 2A). The resulting UV absorption spectrum is also an excellent indicator of effective NP formulations reported by other researchers. 26 In addition, the presence of a single peak depicts the symmetry of the NP produced (Figure 1A). 45 This study demonstrated the ability of EC to form a coating layer around NPs, thereby protecting the NPs from aggregation and ultimately improving physical stability. 46 Figure 2 (A) UV-VIS spectrum of AgNPs-EC, showing the maximum absorbance at 391 nm, which refers to the surface plasmon resonance peak. (B) In vitro Ag release of AgNPs-EC prepared from deionized water (n=3±SD).

Figure 2 (A) The UV-VIS spectrum of AgNPs-EC, showing the maximum absorbance at 391 nm, which refers to the surface plasmon resonance peak. (B) In vitro Ag release of AgNPs-EC prepared from deionized water (n=3±SD).

A basic feature reflected in the behavior of the generated nanoparticles is the release of silver ions from the prepared AgNPs (Figure 2B). ICP-OES was used to detect released silver cations in aqueous media. Initially, silver ions were calculated in AgNPs-EC and found to be 300.50±15.5 µM. The release curve shows the continuous phase for 48 hours. This release performance is due to the limited water solubility of EC in water. 47,48 Previous work showed that compared with other AgNPs prepared using MC, HPMC, and PEG, the previous work of our group showed that the release time of silver cations from AgNPs-EC was extended. 26 It is also interesting that the slow initial release of silver cations is related to the shielding effect of the polymer around the NPs, which affects the entry of water into the NP core. 32 In other words, the cellulose fiber chain may act as a physical barrier to the release of silver cations, as demonstrated before. In addition, the hydroxyl groups in AgNPs-EC can also capture the released silver cations and ultimately limit their release. 49

The physical stability of the developed AgNPs-EC was carried out at two different temperatures for 3 months. Then, the size, charge, and morphology are performed and compared with the newly prepared particles. Morphological observations did not show any significant changes in particle shape. In addition, note the non-significant changes in size and charge, reflecting the higher physical stability of the NPs produced. Therefore, EC plays a prominent role in the formulation and stability of AgNPs (Table 1). Table 1 Characterization of freshly prepared AgNPs-EC and storage after 3 months

Table 1 Characterization of freshly prepared AgNPs-EC and storage after 3 months

The MTT assay was used to study the cytotoxicity of AgNPs-EC in MCF-7 cells after 24 hours. AgNPs-EC was performed using different concentrations (0.1–200 µM). The cells have been incubated for 4 hours at a higher concentration of 200 μM. AgNPs-EC, as determined by MTT assay, is not toxic to cells (Figure 3). According to the findings mentioned by Yingying et al., we used this concentration. 50 They noticed that the toxicity of AgNPs-citrate increases as the concentration increases above 10 µg/mL, while the concentration of 5 µg/mL AgNPs-citrate may increase with low toxicity. Figure 3 The effect of AgNPs-EC on the survival rate of MCF-7. The cell viability of MCF-7 cells treated with AgNPs-EC (0.1–200 μM) was incubated for 24 hours, and then the cytotoxicity was checked using MTT assay.

Figure 3 The effect of AgNPs-EC on the survival rate of MCF-7. The cell viability of MCF-7 cells treated with AgNPs-EC (0.1–200 μM) was incubated for 24 hours, and then the cytotoxicity was checked using MTT assay.

After treatment with AgNPs-EC, we tested cell proliferation. Cell viability was not affected by the increase in AgNPs-EC concentration (Figure 4). Control cells and AgNPs-EC were treated with 5 and 10 μM concentrations for 4 hours, proving that the cells treated with AgNPs-EC proliferated. The enhanced proliferation also emphasizes the inhibitory effect of AgNPs-EC on the differentiation of MCF-7 cells. In 10 μM AgNPs-EC, compared with 5 μM AgNP-EC-treated cells, the proliferation stopped due to the increase of Ag concentration and the cell viability decreased slightly. These findings indicate that the cytotoxic function of AgNPs-EC is mainly related to the release of intracellular Ag. The Ag ions released by 50 nanometer silver particles in aqueous solution have been shown to cause one or two monolayers of oxidized material from their surface, depending on the size of AgNPs-EC. This process is more comfortable at low concentrations and may release high concentrations of Ag, causing cell death. Vice versa, the increase in the concentration of AgNPs is difficult to be ionized. 51 Figure 4 The effect of AgNPs-EC on the proliferation of MCF-7. First, the cell viability of MCF-7 cells was treated with a concentration of 0.1-10 μM.

Figure 4 The effect of AgNPs-EC on the proliferation of MCF-7. First, the cell viability of MCF-7 cells was treated with a concentration of 0.1-10 μM.

MCF-7 cells were pretreated with AgNPs-EC (0.1–10 μM) for 2 hours, and AgNPs-EC had no cytotoxic effect at the used dose. Use a susceptible and accurate quantitative RT-PCR method to measure TNF-α mRNA levels and compare the values ​​with controls. In order to evaluate the effect of protein level suppression on gene expression, TNF-α protein in TNF-α-specific ELISA culture supernatant has been tested. As shown in Figure 5A, the results show that with lower concentrations of AgNPs-EC (0.1, 0.2, 0.5, 5, and 10 μM) (Figure 5B). The production of TNF-α in the supernatant of MCF-7 culture stimulated by 0.1-20 μM AgNPs-EC pretreatment was significantly reduced. In cultures treated with 20 μM AgNPs-EC, the greatest removal was observed. Figure 5 Gene expression and production of TNF-α. (A) The effect of AgNPs-EC on TNF-α gene expression. (B) The effect of AgNPs-EC on TNF-α output in MCF-7 cells.

Figure 5 Gene expression and production of TNF-α. (A) The effect of AgNPs-EC on TNF-α gene expression. (B) The effect of AgNPs-EC on TNF-α output in MCF-7 cells.

Green synthesis is a new field of bio-nanotechnology. Compared with traditional physical and chemical methods, it has economic and environmental advantages. EC and other non-toxic and safe reagents are environmentally friendly and biologically safe. The use of plant extracts to synthesize metal oxide nanoparticles as safe and non-toxic nanoparticles has been studied. 52,53 This study investigated the anti-cancer effects of AgNPs reduced with EC and their effects on TNF-α levels. 54 A sensitive and specific quantitative RT-PCR technique has quantified TNF-α mRNA. The results showed that the level of TNF-α mRNA decreased significantly. The reduction in TNF-α levels may have a positive effect on breast cancer treatment because it is a pro-inflammatory cytokine that is regulated in this type of cancer. It is also considered to be an important executor of tumor induction, promotion, angiogenesis and metastasis. 55 Elevated TNF-α levels are associated with breast cancer recurrence, progression, and metastasis. 56 This means that inhibition of TNF-α can reduce inflammation associated with breast cancer and improve the effectiveness of chemotherapy. It helps prevent or reduce resistance to breast cancer treatment. 3

The increased endogenous TNF-α may be shown to promote tumor invasion by down-regulating the expression of progesterone receptors in breast cancer. 57 When tumor cells are exposed to TNF-α and AgNPs, AgNPs non-specifically bind to TNFR1. Specifically, TNF-α binds to the same receptor to form a TNFR1-TNF-α-AgNPs complex, which enters the cell through endocytosis mediated by the TNF-α receptor. The TNF-α receptor is released and leads to apoptosis. Receptors that are still bound to AgNPs will destroy the contour, molecular weight and characteristics of the receptors, leading to the interruption of their normal cell membrane recirculation pathways, and subsequent reduction of TNFR1 in and within the cell membrane. This molecular mechanism explains how TNFR1 plays a role in enhancing the cellular uptake of AgNPs, because TNF-α-induced apoptosis is reduced. This mechanism explains that the AgNPs-TNFR1 complex can hinder the receptor re-expression pathway on the cell membrane, thereby triggering the reduction of TNF-α signal transduction and its apoptotic effect. 8

In our study, the cytotoxic effect of AgNPs-EC on MCF-7 was evaluated by the MTT assay program after 2 hours. Different concentrations of AgNPs-EC did not affect cell viability. These findings indicate that the cytotoxic function of AgNP-EC is almost ignored for normal cells and can be considered safe. It only works by reducing TNF-α, which is responsible for proliferation, progression, and tumor metastasis. However, the mechanisms related to the cytotoxicity of AgNPs have not been fully understood. In order to explain the cytotoxicity, it was agreed that the interaction of AgNP-EC with the thiol group of the inner mitochondrial membrane occurred. They proposed that this would lead to depletion of the antioxidant defense system, leading to the production of ROS. The result of ROS accumulation is inflammation. Mitochondrial destruction is triggered by the inflammatory response of cells, which can lead to apoptotic factors that induce cell death. 58

AgNPs produce different biochemical pathways, including ROS-mediated mitochondrial dysfunction, DNA damage and apoptosis, which are related to the enhanced anti-cancer activity in MCF-7 cells. 53,59 The results we obtained in this study are consistent with the results of the following research researchers in previous exams. Mugade et al. used bioengineering (Mannan sulfate-terminated AgNPs) to down-regulate the expression of TNF-α and IL-6 in rats. 60 Wong et al. also studied the anti-inflammatory activity of AgNPs and found that AgNPs can reduce the production of TNF-α. 61 In addition, this study was mediated by Liu et al. and demonstrated that dendrimer-encapsulated AgNPs (AgNPs-DNC) can be used in Inhibit the production of TNF-α and IL-6 in vitro. 62 These findings indicate that the potential anticancer activity of AgNP-EC may be related to apoptosis by inhibiting the activation of mRNA and nuclear translocation in MCF-7 cells and the protein expression of TNF-α. AgNPs-EC has a strong effect on inflammation and TNF-α promoted toxicity.

On the other hand, the toxic effects of AgNPs have been confirmed in a number of in vivo studies. This effect is related to many variables, such as size, concentration, route of administration, and internal use. AgNPs are considered a double-edged sword. In this study, it was found that the concentration of AgNPs-EC used was non-toxic to normal cells, which is a good sign of the safety of these cells. According to Fehaid and Taniguchi, these prepared nanoparticles are not toxic to normal cells due to their relatively large diameter. The cytotoxicity of AgNPs is related to size. The small size of about 10 nm is easily ionized and releases Ag which is toxic to cells. However, the cell-based absorption of the 150 nm particles prepared in this study can occur through the endocytosis of their spheres. When contained in the endosome and not easily ionized, they are not easily touched, which results in lower cytotoxicity. 63

However, in order to determine their anti-cancer activity potential, clinical studies are needed in the future. The biosynthetic AgNPs-EC was studied for the treatment of cell line breast cancer (MCF-7). Studies have shown that these AgNPs-ECs are non-toxic and successfully penetrate cancer cells within the studied concentration range (1-100 µg/mL). Previous studies have also discussed that AgNPs can be used as inert drug preparation platforms and active reagents that can affect the function of the cell system, combining with endosomes, exosomes and lysosomes according to their size and possible charge. 64 These studies were supported by Hussain et al.'s studies on the toxicity of AgNPs (15 and 100 nm) in BRL 3A of rat liver cells. In this type of cell, they discovered the toxicity of AgNPs. They also determined that in cells treated with AgNPs, mitochondrial function was significantly reduced at 5-50 μg/mL. 65

AgNPs-EC acts as an inhibitor of TNF-α to a considerable extent to support theories about inhibiting cancer through physiological and biochemical signals. These findings play an important potential role in revealing new ways of cancer prevention and care.

The author thanks the Deputy Minister of Research and Innovation of the Ministry of Education of Saudi Arabia for funding this research work through the project number QU-IF-1-2-1. The author would also like to thank Qasim University for its technical support.

The author declares that there is no conflict of interest in this work.

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