Cytotoxicity of synthesized silver nanoparticles on breast cancer cells

. Breast cancers are becoming harder to treat due to the acquisition of chemo-drug resistance. In this study, silver nanoparticles (AgNPs) were synthesized using reducing agent NaBH 4 , where resulting nanoparticles were characterized using UV-vis spectroscopy, FTIR spectroscopy, SEM, and DLS. Cytotoxicity of synthesized AgNPs was evaluated against MCF-7, MCF-7-CR, and MDA-MB-231 using MTT assays. NaBH 4 -reduced AgNPs were unstable as a colloidal system, with zeta potential noted to be around -21 mV and a polydispersity index of around 15%, making them highly prone to aggregation. However, AgNPs significantly reduced the cell viability of MCF-7 breast cancer cell lines, while slight toxicity was seen in multi-drug resistant breast cancer cells MCF-7-CR and MDA-MB-231 at 10 µM.


Introduction
Multi-drug resistance (MDR) is a major problem in cancer, especially breast cancer chemotherapy as it makes administering single or multiple drugs harder.Most anticancer drugs also have a non-specific cytotoxic activity towards surrounding normal cells, which can cause further complications [1].
Metallic nanoparticles have been used in cancer research and are documented to induce toxicity against MDR cancer cells [1].Among them, silver nanoparticles (AgNPs) can induce cytotoxicity in cancer cells via a "Trojan-horse" type mechanism, where internalized AgNPs induce cell death [2].AgNPs localize in the cytosol, mitochondria, and endoplasmic reticulum, resulting in enzymes and growth factor inhibition related to angiogenesis and cell proliferation [3,4] AgNPs were also associated with apoptosis as oxidative stress generation and DNA damage were seen after administration to cancer cells [5][6][7].
Cytotoxic AgNPs can be synthesized chemically using a combination of reducing agents to initiate redox reactions between silver ions and stabilizers to stabilize the formation of silver nucleates from degradation [8].Chemical synthesis can result in high AgNPs yield and ease of parametric customization, however, most reducing agents used can be toxic and have adverse effects [9,10].'Green' chemistry could be applied to chemical synthesis by using phytochemicals or biological system-mediated synthesis for synthesizing bioactive compound-conjugated AgNPs capable of treating cancer cells [11][12][13].While 'green' chemistry can be a non-toxic synthesis alternative, their overreliance on natural product extracts and living organisms results in a high degree of inconsistency as well as industrial scalability limitations in terms of both economic and operations [14,15].

Silver Nanoparticle (AgNPs) Synthesis
20 µL of freshly made 5 mM NaBH4 was added to 20 mL of 1 mM AgNO3 dissolved in deionized water (DI H2O).Upon addition, the mixtures were stirred for 30 minutes at 350 RPM using a stirring hotplate (Fisher Scientific, USA), before being stored at 4°C.

UV-Visible (UV-vis) Spectroscopy
100 µL of synthesized AgNPs colloidal samples were loaded onto a transparent 96-well plate for analysis.The plate was read using the Infinite M Plex plate reader (Tecan, Swiss) with sweeping absorbance ranging from 250-800 nm wavelength.

Scanning Electron Microscopy (SEM) Observation
Freshly made 20 µL of synthesized AgNPs were dropped on microscope slides and freezedried using ScanVac CoolSafe 110-4 Basic 4lt Freeze Dryer and RV5 Rotary Vane Vacuum Pump.Microscope slides were then sputtered with platinum ion coating using SPT-20 (Coxem, Korea) and mounted to Tescan Vega 3 SEM (Tescan, Czech) to be visualized under 15000x magnification.Images were analyzed using ImageJ software.

Dynamic Light Scattering (DLS) Analysis
Synthesized AgNPs suspension was used to measure zeta potential using Litesizer 500 (Anton Paar, Germany).

MTT Cell Viability Assay
Non-MDR breast cancer cell line MCF-7, cisplatin-resistant breast cancer cell lines MCF-7-CR, and MDR MDA-MB-231 passages p12 were seeded in transparent 96-well plates at a calculated 15000 cells/well.Cells were grown in DMEM completed with 10% FBS and incubated at 37°C with 5% CO2 in CelCulture® CO₂ Incubator Model CCL-170T-8 (Esco, Singapore) until 70% confluency was observed.Cells were treated in DI H2O-diluted 1 mM AgNP at final concentrations 2, 4, 6, 8, and 10 µM.After 24h treatment, cells were treated with 0.5 mg/mL MTT for 2h. 100 µL DMSO was added to cells and results were then read with the Infinite M Plex plate reader at 570 nm wavelength.

Results and Discussion
AgNPs synthesized nanoparticles were characterized using UV-vis spectroscopy, where max absorbance for AgNPs was found at around 380-420 nm (Fig. 1.).Plasmonic spectral peak was detected specifically at 400 nm.UV-vis spectral peaks of AgNPs were associated with the presence of AgNP in colloid form and the peak and amplitude shifting were recorded to be correlated to the size and shape morphology of AgNPs [26].Synthesized AgNPs were characterized using FTIR spectroscopy and were found to have spectral peaks at 2669, 2354, 2042, 1753, 1283, 800, and 732 cm -1 wavenumbers (Fig. 2

.).
The strong peaks at 1283, 800, and 732 cm -1 corresponded to surface chemical fingerprint of nanoparticles, which was confirmed to be highly similar to peaks for silver nitrate [27] Another small peak at 2354 cm -1 also corresponded to B-H stretching, signifying traces of NaBH4 found around the surface of AgNPs [28].After comparison with reference H2O FTIR for background noise and control, no IR spectra for synthesized AgNPs were found to be associated with O-H stretching and H-O-H bending [29].

Fig. 2. FTIR spectra characterization of synthesized AgNPs reduced in NaBH4.
AgNPs were calculated to have a mean particle size of 680 ± 188 nm.AgNPs were shaped irregularly and unevenly, with some shaped relatively spherical and seemed to fuse in an aggregate (Fig. 3.).The polydispersity index of AgNPs and mean zeta potential were also measured to be 15.24% and -21.35 mV respectively.The measured zeta potential and polydispersity index suggested that the synthesized nanoparticles were not stably dispersed in water, as the electrokinetic potential between the Ag solid and the ionic solvent could not form a large enough electrostatic and steric repulsion layer [30,31].Further, the highly irregular morphology suggested the AgNPs had a high flocculation and agglomeration rate, noting that NaBH4 alone was not adequate to form stably dispersed AgNPs colloid despite the confirmed presence of synthesis.After stabilization using PVA, Roto et al. reported stable NaBH4-synthesized AgNPs colloid, parametric value shifting in UV-vis spectra, as well as changes in size-shape morphology [32].AgNPs were tested for cytotoxicity against MCF-7, MCF-7-CR, and MDA-MB-231 cells; where MCF-7-CR cells are resistant to the FDA-approved drug Cisplatin [25] while MDA-MB-231 cells are metastatic-stage, drug-resistant mammary cancer.Synthesized AgNPs were found to exhibit a dose-dependent effect where AgNPs concentrations were proportionally related to the viability reduction of both cell lines (Fig. 4.).AgNPs were able to significantly lower cell viability to about 20% for MCF-7 at 10 µM, while cell viability was significantly reduced to 10% for MCF-7-CR.In the case of the MDA-MB-231 cell line, AgNPs were able to induce cytotoxicity in cells at 10 µM albeit not as potent as in MCF-7 and MCF-7-CR cells to only about 5% cell death.The result indicated that AgNPs-induced cytotoxicity was not as effective in MDR phenotype breast cancer cells as compared to non-MDR breast cancer MCF-7 cell lines.While the mechanism of action of AgNPs toxicity is not fully understood, it is widely known that stable AgNPs, are associated with necrosis and lipid peroxidation [33].In some cases, AgNPs could be degraded into Ag + ions by lysosomes and produce oxidative stresses in the nucleus and mitochondria, thereby promoting apoptosis induction in certain cells [2,34].In one study, Kaur et al. found that NaBH4-reduced AgNPs were not as toxic as tannic acid-reduced AgNPs when treated on A431 skin carcinoma and A549 lung cancer, with cell viability for NaBH4 reaching 95-90% at 100 µg/mL, while tannic acid resulted in cell viability of 75-60% [18].

Conclusion
Nanoparticles, especially AgNPs can be used in the medical field of study as a form of therapeutics.In this study, AgNPs were synthesized using reducing agent NaBH4 and characterized to have a large particle size as well as low polydispersity index and zeta potential, making the nanoparticles very prone to agglomeration.Despite this, synthesized AgNPs were able to exhibit cytotoxicity even in the MDR phenotype breast cancer cell line, suggesting that some AgNPs were successfully taken up by MCF-7, MCF-7-CR, and MDA-MB-231 cells.Further studies should be conducted with the addition of stabilizing agents to regulate the morphology and charge of AgNPs to influence cytotoxicity in breast cancer cells.Investigation of the underlying mechanism of MDR bypassing by AgNPs is also crucial.
We would like to thank Sunway University for their support.