In vitro anti-cancer and antimicrobial e ﬀ ects of manganese oxide nanoparticles synthesized using the Glycyrrhiza uralensis leaf extract on breast cancer cell lines

: In this study, we evaluated the antiproliferative and apoptotic properties of Pluronic-F127-containing manganese oxide nanoparticles (PF-127-coated Mn 2 O 3 NPs) derived from the leaf extract of Glycyrrhiza uralensis (GU) on breast adenocarcinoma, MCF7, and MDA-MB-231 cell lines. The leaf extract of GU contains bioactive molecules that act as a reducing or capping agent to form Mn 2 O 3 NPs. Various analytical techniques were used to characterize the physiochemical properties of PF-127-coated Mn 2 O 3 NPs, including spectroscopy (ultralight-Vis, Fourier transform infrared, photoluminescence), electron microscopy ( ﬁ eld emission scanning electron microscopy and transmission electron microscopy), X-ray di ﬀ raction (XRD), electron di ﬀ racted X-ray spectroscopy (EDAX), and dynamic light scattering. The average crystallite size of Mn 2 O 3 NPs was estimated to be 80 nm, and the NPs had a cubic crystalline structure. PF127-encapsu-lated Mn 2 O 3 NPs signi ﬁ cantly reduce MDA-MB-231 and MCF-7 cell proliferation, while increasing endogenous ROS and lowering mitochondrial matrix protein levels. DAPI, EtBr/AO dual staining, and Annexin-V-FITC-based ﬂ ow cytometry analysis revealed that PF127-coated Mn 2 O 3 NP-treated breast cancer cells exhibit nuclear damage and apoptotic cell death, resulting in cell cycle arrest in the S phase. Furthermore, PF127-encapsulated Mn 2 O 3 NPs show strong antimicrobial e ﬃ cacy against various strains. As a result, we can conclude that PF127-coated Mn 2 O 3 NPs may be e ﬀ ective as future anticancer agents and treatment options for breast cancer.


Introduction
Nanotechnology involves the science, engineering, and manipulation of nanoscale materials and devices, aiming to develop improved characteristics and innovative properties.Nanoparticles (NPs) are extensively employed and ratios, have potential applications in biological, catalytic, mechanical, and electroconductivity.Metal oxide NPs have higher surface area and volume ratios than bulk materials, making them suitable for antibacterial and cancer treatments [2].
Green nanotechnology is a key component of clean technologies intended to preserve the environment and transform supplemental bioactive components into more economical and environmentally beneficial green nanomaterials [3].Green chemistry utilizes plant extracts to reduce and stabilize manganese metal into NPs, offering scalability, medical applications, and biocompatibility [4,5].Eco-friendly, sustainable, cost-effective, and simple, this process outperforms conventional chemical-based techniques [6].
Green nanotechnology focuses on the environmentally friendly, sustainable development and utilization of nanomaterials, minimizing environmental and health risks while maximizing benefits.It involves eco-friendly synthesis methods, renewable materials, and environmentally benign approaches throughout their lifecycles [7].Metallic NPs, including silver, gold, copper, iron oxide, and zinc oxide NPs, have attracted considerable attention in nanotechnology due to their unique properties and versatile applications [8].These NPs possess distinctive physicochemical properties, such as a large surface-to-volume ratio, quantum confinement effects, and localized surface plasmon resonance, which make them attractive for a wide range of applications [9].Nanotechnology, including green and metallic NPs, advances pharmaceutical and biomedical fields.It improves drug delivery, imaging, diagnostics, therapeutics, tissue engineering, and cancer treatment.Further research and development can improve healthcare and address biomedical challenges [10].
Green synthesis of Mn 2 O 3 NPs offers several advantages over traditional physical and chemical synthesis methods.
Here are some green synthesis advantages: green synthesis methods utilize natural and renewable resources, such as plant extracts, as reducing and stabilizing agents.This reduces the dependence on hazardous chemicals and minimizes the generation of toxic by-products, making it an ecofriendly approach [11].Green synthesis methods often use low-cost starting materials and simple reaction conditions, making the overall process more cost-effective than traditional methods requiring expensive reagents and complex equipment [12].Green synthesis methods generally operate under ambient or mild reaction conditions, such as room temperature and atmospheric pressure.This saves energy and preserves the bioactivity and stability of synthesized NPs [13].Green-derived Mn 2 O 3 NPs are typically made with biocompatible and biodegradable materials, making them suitable for various biomedical applications.These NPs can be used in drug delivery, imaging, diagnostics, and therapeutic applications with reduced toxicity and improved biocompatibility [14].Green synthesis methods provide tunable properties for Mn 2 O 3 NPs by adjusting reaction parameters or using natural reducing agents.This allows for tailored properties for specific applications like catalysis, sensors, and optoelectronics [15].Green-synthesized metal oxide NPs often exhibit enhanced stability and functionality due to natural capping agents or biomolecules on their surfaces [16].
Metal NPs and oxides offer valuable biomedicine benefits due to their large surface area, reduced size, and lightabsorbing properties.They are used for their antimicrobial, antitumor, anti-larvicidal, and antihyperglycemic properties.[17].Mn oxides have drawn particular interest among other 3D transition metal oxides because of their numerous compositional and structural variations, including MnO, Mn 5 O 8 , Mn 2 O 3 , MnO 2 , and Mn 3 O 4 [18].Their physicochemical characteristics and several applications in biosensory, catalysis, ion exchange, molecule sorption, energy storage, and drug delivery have all been investigated [19,20].Additionally, Mn oxides often have lower toxicity levels than other compounds, which form the basis for most NPs.They are also more environmentally friendly, stable, have higher specific capacitance, and are more economical [21].
Pluronic F-127 (PF-127) is an amphiphilic triblock copolymer consisting of a central hydrophilic polyethylene oxide chain surrounded by two hydrophobic polypropylene oxide chains [22].Due to its strong dissolving power, biocompatibility, reverse gelation, and low toxicity, PF-127 is considered an excellent medium for effective drug delivery via a variety of parenteral and non-parenteral routes [19].Extensive research on the functional properties of these polymers in recent years has led to the development of several systems widely used as drug delivery and release mechanisms for proteins, nucleic acids, and peptides [23].
Herbal extracts contain phytochemicals such as alkaloids, polyphenols, tannins, terpenoids, and alcohol, which stabilize and reduce NPs.These plant extracts inhibit microbial pathogens and are used in green Mn 2 O 3 NP synthesis [24].Lemon, kalopanax, Syzygium, Phyllanthus, ananas, dittrichia, and yucca extracts are used in synthesis techniques for green Mn 2 O 3 NPs [25].However, the leaves from the plant Glycyrrhiza uralensis (GU) have not been employed for green Mn 2 O 3 NP synthesis.A perennial herb belonging to the genus Glycyrrhiza, licorice is mostly found in China [26].Licorice is widely used in various industries, including food, tobacco, cosmetics, health care, and pharmaceuticals [27].With over 400 chemicals identified, it has strong antitussive, asthmatic, hepatoprotective, anti-inflammatory, antiviral, anti-ulcer, and anti-diabetic properties [23].
Licorice's underground component is used in China for various illnesses, while its aerial components, such as leaves, have significant nutritional content [28].
The current study describes the environmentally friendly synthesis and characterization of PF-127-encapsulated Mn 2 O 3 NPs with the GU leaf extract, as well as their biomedical applications, including antimicrobial and anticancer activity against MDA-MB-231 and MCF-7 breast cancer cells.

Preparation of the GU leaf extract
About 10 g of fresh GU leaves were mixed with 100 mL of ethanol and boiled for 20 min at 80°C.The obtained extract was filtered using filter paper.The liquid was collected in an Erlenmeyer flask and stored at 37°C for later use.

Preparation of PF-127-encapsulated Mn 2 O 3 NPs
To prepare PF-127-encapsulated Mn 2 NO 3 NPs, 100 mL of the GU leaf extract was mixed with 0.1 M of manganese(II) nitrate tetrahydrate (Mn(NO 3 ) 2 •4H 2 O) and 0.5 g of PF-127.The resulting green-yellow homogeneous mixture solution was continuously agitated at 80°C for 5 h.The white precipitates were allowed to dry for 1 h at 120°C.The powdered PF-127-coated Mn 2 O 3 NPs were annealed at 800°C for 5 h before collection for further use.

Characterization of PF-127-coated Mn 2 O 3 NPs
A Bruker-AXS D5005 X-ray diffractometer was used to characterize PF-127-coated Mn 2 O 3 NPs.The Cu-K radiation with a wavelength equal to 0.1541 nm was used for the analysis, which was scanned at an angle of 2°between 20°and 90°.
The average particle size of the NPs was estimated by dynamic light scattering (DLS).The experiment was carried out with NPs suspended in deionized distilled water at a scattering angle of 90°and a temperature of 25°C.The size and surface morphologies of PF-127-coated Mn 2 O 3 NPs were determined by field emission scanning electron microscopy (FESEM) on a Hitachi s-4800II equipped with electron diffracted X-ray spectroscope (EDAX) to study the elemental arrangement of the formulated NPs.A photomicrograph was taken at 20 kV.To study the NP morphology further, a transmission electron microscope (TEM; Jeol Jem-2010F) was employed.In brief, the NPs were smeared on a copper grid and illuminated with electronic radiation in a vacuum.After passing through the material, an electron beam captured microphotographs.The functional groups in the PF-127-encapsulated Mn 2 O 3 NPs were investigated using Fourier transform infrared (FTIR) spectroscopy (NicoletiS50).The reflectance method was used to identify the IR spectrum.The NPs were ground with KBr in a 1:100 ratio and flattened into discs to make potassium bromide (KBr) discs.The discs were scanned in the 400-4,000 cm −1 range directly into the spectrometer.To confirm the formation of the NPs and identify the surface plasmon resonance peak, the NPs were subjected to doublebeam ultraviolet-visible (UV-Vis) spectroscopy (Shimadzu UV-2550, USA).The nanomaterials were tested in the wavelength range of 1,200-200 nm three times to determine the mean absorbance of the NPs.Photoluminescence (PL) spectroscopy (Roithner Laser Technik) was used to assess the optical properties of the NPs.The spectra were examined at exc = 470 nm using the 350-550 nm spectral area.The experiment was run three times, and the energy band gap was calculated by plotting Tauc with the following equation: where h is Planck's constant; A is the constant proportion; α is the absorption coefficient; v is the vibration frequency; E g is the band gap; n is the sample transition nature [29].
2.5 Biological studies

Analysis of apoptotic cells
The apoptotic efficiency of PF-127-coated Mn 2 O 3 NPs against MDA-MB-231 and MCF-7 cells was examined using the dual ethidium bromide/acridine orange staining method.Cells were exposed to IC 25 and IC 50 NPs for 24 h, treated with an acridine orange/ethidium bromide solution for 5 min, and rinsed with phosphate-buffered saline (PBS) before being examined under an EVOS Cell Imaging System (Thermo Fisher Scientific Inc., MA, USA) [30].

JC-1 staining
Breast cancer cells were treated with green-synthesized Mn 2 O 3 NPs, and their mitochondrial matrix protein (MMP) status was evaluated using JC-1 fluorescent staining.Both cells were separately cultured at a density of 2 × 10 5 per well in a 6-well plate for 24 h, then treated with NPs of IC 25 and IC 50 values.Later, 1 µg•mL −1 of JC-1 fluorescent dye was loaded into the wells for 20 min and then washed in PBS before being examined under a fluorescence microscope (Thermo Fisher Scientific Inc.).

DCFH-DA staining
Endogenous reactive oxygen species (ROS) accumulation in both treated and control breast cancer cells was measured using DCFH-DA fluorescent staining.Both cells were cultured in a 24-well plate for 24 h before being administered with NPs of IC 25 and IC 50 values for 24 h.The fluorescent stain DCFH-DA was then applied to each well for 1 h.The level of endogenous ROS was then measured in both control and treated cells using a fluorescent microscope (Thermo Fisher Scientific Inc., MA, USA).

Analysis of apoptosis by flow cytometry
The effect of green-synthesized Mn 2 O 3 NPs on apoptosis in breast cancer cells was studied with flow cytometry.On a 6-well plate, both cells were cultured separately for 24 h.They were then exposed for 24 h to the IC 25 and IC 50 values of green-synthesized Mn 2 O 3 NPs.Apoptosis was measured in control and treated cells by the Annexin V-FITC/PI-Apoptosis Detection Kit.After the cells were harvested, they were studied using flow cytometry according to the manufacturer's instructions (Abcam, USA).

Cell cycle analysis
Cells were rinsed with a buffered solution before being treated with ethanol for 12 h.The cells were then stained for 30 min with 300 µL of a staining solution containing 100 L of propidium iodide (PI), 0.08 mg•mL −1 of a proteinase inhibitor, and 0.5 mg•mL −1 of RNase.Flow cytometry was used to measure PI fluorescence in relation to DNA.The MultiCycle program (Phoenix Flow Systems, USA) was used to examine nuclei percentages at various stages of the cell cycle.

Statistical analysis
All experiments were run in triplicate, and the data were statistically analyzed using a one-way ANOVA and a posthoc Tukey post-test.The values are presented as mean ± standard deviation, with statistical significance set at P < 0.05.

Characterization of green PF-127
Mn 2 O 3 NPs

UV-Vis spectrometry analysis
The optical properties of PL127-coated Mn 2 O 3 NPs were investigated using a UV-visible (UV-Vis) spectrometer, and UV-Vis wavelengths ranging from 200 to 1,100 nm were recorded.The UV and optical spectrum of PL-127-encapsulated Mn 2 O 3 NPs is depicted in Figure 1a.When electrons are excited from the filled to the empty band, Mn 2 O 3 NPs' absorbance edges are visible at 232 and 394 nm (Figure 1a) [31].

FTIR spectrum analysis
The functional groups present in GU were identified and their function in the synthesis of PL127-coated Mn    (622) of Mn 2 O 3 NPs are represented by the peaks at 22.8°, 32.7°, 37.99°, 44.96°, 49.12°, and 55.02°, respectively, in the XRD spectrum.The hkl planes are well-indexed to a cubic structure in comparison to the typical peaks of pure Mn 2 O 3 (JCPDS 41-1442) [35]:

X-ray diffraction (XRD) analysis
where the X-ray wavelength (1.5406), Bragg's diffraction angle, and the angular peak width at half maximum (rad) are all given.
Green PL-127-coated Mn 2 O 3 NPs have an average particle size of 80 nm.Following a successful synthesis, the average particle size of the NPs was estimated using the Debye-Scherrer equation and an examination of the material's crystallinity [36].

DLS study
The hydrodynamic size of PL127-coated Mn 2 O 3 NPs was calculated using DLS to determine the particle size, as Effect of NPs synthesized using the GU leaf extract on breast cancer  7 shown in Figure 3b.Because a water medium encircled the PL127-coated Mn 2 O 3 NPs, which were measured at 195.50 nm in size, the DLS particle size was larger than that of the XRD results.This sizing is known as the hydrodynamic size.

Antimicrobial activity of Mn 2 O 3 NPs
The antibacterial activity of PL127-coated Mn 2 O 3 NPs was assessed against Gram-negative bacterial strains (K.pneumonia, S. dysenteriae, and E. coli) and the fungus C. albicans, as depicted in Figure 4a.Amoxicillin, an antibiotic, and green PL127-coated Mn 2 O 3 both exhibited substantial antimicrobial properties.In comparison to amoxicillin, the PL127-coated Mn 2 O 3 NPs demonstrated larger zones of inhibition (Figure 4b).Additionally, the antimicrobial activity improved as the nanoparticle concentration increased.

Analysis of apoptotic nuclear changes by DAPI staining
DAPI staining was used to analyze the apoptosis in the control and treated breast cancer cells.As demonstrated in Figure 7, the control cells exhibited fewer fluorescent cells, which indicates viable cells without or with less apoptosis.However, both cells exposed to (IC 25 , IC 50 ) green-synthesized PL127-coated Mn 2 O 3 NPs demonstrated increased blue fluorescence, indicating the occurrence of higher apoptotic cell death with increased structural damages, nuclear injury, and apoptotic body formations in both cells (Figure 7b and c).

MMP analysis
The PL127-loaded Mn 2 O 3 NP-induced changes in breast cancer cells' MMP were examined by JC-1 staining, and the results are shown in Figure 8.The untreated control cells exhibited red fluorescence, which reveals intact MMP.However, both cells, which were exposed to the (IC 25 , IC 50 ) green-synthesized PL127-coated Mn 2 O 3 NPs, produced increased green fluorescence, which indicates that PL127-coated Mn 2 O 3 NP treatment could reduce MMP levels of breast cancer cells (Figure 8).

Analysis of ROS accumulation
The role of ROS generation in green-synthesized PL127-coated Mn 2 O 3 NPs-induced apoptosis in breast cancer cells is of utmost importance.Figure 9 depicts the effect of greensynthesized PL127-coated Mn 2 O 3 NPs on endogenous ROS accumulation in breast cancer cells.As displayed in Figure 9, the control cells exhibited poor green fluorescence, which reveals less ROS generation; whereas, the MDA-MB-231 and MCF-7 cells, which were exposed to (IC 25 , IC 50 ) greensynthesized PL127-coated Mn 2 O 3 NPs, demonstrated increased green fluorescence, which reveals increased ROS levels.

Analysis of apoptosis by flow cytometry
In the current study, the Annexin V/PI assay, which assesses apoptosis, was utilized to further study the potential cell death mechanisms of the breast cancer cells following treatment with the green-synthesized PL127-coated Mn 2 O 3 NPs.The percentage of apoptotic cells in treated breast cancer cells was investigated using flow cytometry analysis (Figure 10).The results of flow cytometry indicated that treatments with (IC 25 , IC 50 ) green-synthesized Effect of NPs synthesized using the GU leaf extract on breast cancer  9 PL127-coated Mn 2 O 3 NPs considerably augmented the apoptotic cell proportions in both cells.Both cells showed higher apoptosis after treatment with PL127-coated Mn 2 O 3 NPs (Figure 10).

Cell cycle analysis
Flow cytometry was used to detect cell cycle inhibition in control and treated breast cancer cells (Figure 11).Following  treatment with green-synthesized PL127-coated Mn 2 O 3 NPs, both cells showed an increase in the S-phase cell proportion.Furthermore, after exposure to Mn 2 O 3 NPs, cell proportions in the G1 phase were significantly reduced in both cells.These results show that treatment with PL127-coated Mn 2 O 3 NPs induces cell cycle arrest in the S phase in breast cancer cells (Figure 11).

Discussion
Cancer, one of the most debilitating illnesses in the world, leads to the death of millions of individuals in underdeveloped nations.It has been documented that oxidative stress is a crucial factor in the emergence and progression of multiple diseases that affect essential organs, especially cancer [37].Breast cancer ranks among the most often diagnosed types of cancer.Breast cancer can be treated with several treatment methods, including surgery, radiation therapy, and chemotherapy.Unfortunately, these treatments can have negative impacts on healthy cells [38].The existing chemotherapeutic drugs have a very constrained therapeutic range, limited solubility, and are hazardous to healthy tissues [39].In this aspect, polymer-based drug delivery systems can provide highly localized drug concentration in tumorous regions with no detrimental impact on healthy cells [40].
The antimicrobial mechanism of NPs can be ascribed to an electrochemical action model that permeates and damages the cell walls of the microbes, thereby inhibiting their growth [41].The remarkable antimicrobial activity of Mn 2 O 3 NPs is attributable to electron transport in an oxidized and reduced state, and NPs are believed to have inactivated bacterial enzymes, producing H 2 O 2 and resulting in bacterial cell death [42].Moreover, their physical properties including size and morphology in addition to the functionalization with the plant extract molecules are also equally responsible for the high antimicrobial effect displayed by NPs.The findings demonstrate that Mn 2 O 3 NPs have outstanding antimicrobial characteristics and have the potential to provide effective targeted delivery to the microbes for maximized bactericidal effect [43].Factors influencing the biological activity of inorganic NPs include size distribution, morphology, surface charge,  surface chemistry, and capping agents.These variables can improve targeting efficiency, safety, and effectiveness in biomedical applications like drug delivery, imaging, diagnostics, and therapy [44].
The MTT technique, which relies on the formation of formazan complex via the reduction of MTT or other tetrazolium salts, is a cytotoxicity assay to assess the cell death rate.Numerous studies have demonstrated that the elimination of free radicals promotes the anti-cancer activity exerted by metallic NPs [45].Oxidative stress induced by elevated ROS levels inside the cells ultimately results in programmed cell death.Additionally, damaged mitochondrial membranes caused by high ROS levels have been associated with cell death.Additionally, PF-127 that was utilized to encapsulate the NPs has been demonstrated to interact with cancerous cells, thereby chemo-sensitizing them [46].
Green NPs cause cell death through loss of membrane integrity due to the release of Mn 4+ ions, which trigger ROS accumulation.This further enhances DNA damage, mitochondrial dysfunction, and apoptotic cell death due to ROSinduced oxidative stress [47].The initiating factors and hallmarks of apoptosis are shrinkage and detachment of cells, plasma membrane protrusions, and the development of apoptotic bodies and nuclear fragmentations.Quercetinloaded AuNPs effectively induce higher apoptotic incidences in breast cancer cells, supporting the ability of green-synthesized Mn 2 O 3 NPs to induce apoptosis [48].Anticancer drugs activate apoptosis via the mitochondrial pathway, with altered MMP, which is a key indicator of apoptosis [49,50].The treatment with MnO 2 NPs led to decreased MMP levels in breast cancer MCF-7 cells, consistent with the current findings [51].
ROS have long been linked to tumor growth due to their influence on cellular proliferation and chemotherapy resistance.Cellular damage or death, on the other hand, can result from either an excess of ROS accumulation or a compromised cell's ability to counteract ROS.As a result of oxidative stress, tumor cells die due to their excessive ROS accumulation [52].Overproduction of ROS is a common effect of chemotherapeutic drugs that might initiate apoptosis.These findings evidence that treatment with Mn 2 O 3 NPs induces oxidative stress in breast cancer cells by increasing ROS.This study's findings were supported by a previous study demonstrating increased ROS accumulation in MDA-MB-231 and MCF-7 cells after carbon nanoparticle treatment [53].
Previously, MnO 2 NPs were synthesized from the Saraca asoca leaf extract (SA-MnO 2 NPs), which were found to be significantly cytotoxic against MDA-MB-231 and MCF-7 cells [54].Based on an evaluation of green-derived MnO 2 NPs using Viola betonicifolia leaf extract against MCF-7 breast cancer cells, up to 96% inhibition was observed in earlier studies [55].A study found that green-fabricated MnO 2 NPs derived from the Terminalia chebula fruit extract inhibited MCF-7 breast cancer cells [56].In earlier studies, green MnO 2 NPs prepared using the Gmelina arborea extract showed higher antitumor activity.Further, cells treated with GAE-coated MnO 2 NPs showed normalized cellular morphology, indicating improvement in antitumor activity [57].In comparison with previous studies, our results described that the GU leaf extracts were used to synthesize Mn 2 O 3 NPs that were highly cytotoxic to MDA-MB-231 and MCF-7 cells.In addition to increased ROS accumulations and decreased MMP levels, DAPI, dual staining, and flow cytometry demonstrated increased apoptosis and cell cycle inhibition.
Apoptosis, a critical component in the regulation of cell death, plays a major role in determining cancer cell growth and death.The avoidance of apoptosis is a crucial factor in tumor growth and the emergence of resistance to therapies.It had already been shown that suppressing apoptosis enhances cancer progression [58].The flaws in apoptotic mechanisms trigger cells to proliferate continuously, which is a hallmark of tumors, and tumors are often generated by colonies of immortal cells that have mutated to evade apoptosis.Thus, inducing cancer cells to self-destruct via apoptosis is considered a promising strategy for tumor eradication.An earlier study done by Moghaddam et al. [59] highlighted that Mn 2 O 3 NPs effectively induced apoptosis cell death in MCF-7 cells, which supports the current findings.The cell cycle consists of interphases (G1, S, and G2) and mitosis (M).In the G1 phase, cells grow and make RNA and proteins for DNA synthesis [60].In the S phase, DNA replicates, and the G2 phase involves protein synthesis.The M phase divides the nucleus and cytoplasm.Controlling the tumor cell cycle is crucial for anticancer therapies.A study using green-synthesized silver NPs supports the findings [61].

Conclusions
Eco-friendly P127-coated Mn 2 O 3 NPs coated with the GU leaf extracts have been synthesized and characterized for their antimicrobial and anticancer activities.The NPs have an average crystallite size of 80 nm and exhibit high antimicrobial efficacy against various bacterial strains and the fungal Candida albicans.The green-formulated PL-127-coated Mn 2 O 3 NPs exhibited a significant cytotoxic effect on breast cancer cells.Increased ROS levels and reduced MMP levels were observed in PL-127-coated Mn 2 O 3 NP-treated breast cancer cells.The findings of dual staining, DAPI and Annexin V-FITC/PI staining, revealed increased apoptosis in breast cancer cells after treatment with greensynthesized PL-127-coated Mn 2 O 3 NPs.Furthermore, flow cytometry analysis revealed cell cycle inhibition in breast cancer cells.Thus, it was evident that green-synthesized PL-127-coated Mn 2 O 3 NPs possess significant antimicrobial and anticancer treatment options for breast cancer.Future studies will concentrate on using newly created PL-127-coated Mn 2 O 3 NPs for nanodrug delivery platform design and development.

2. 6 . 4 4′, 6 -
Diamidino-2-phenylindole (DAPI) staining DAPI staining was used to assess nuclear DNA damage in breast cancer cells treated with PL-127-encapsulated Mn 2 O 3 NPs.Cells were seeded, incubated at IC 25 and IC 50 concentrations, and stained with 200 ng•mL −1 DAPI fluorescent dye for 15 min.The EVOS Cell Imaging System was used to investigate nuclear damage in treated cells (Thermo Fisher Scientific Inc.).

Figure 2 :
Figure 2: (a and b) FESEM and TEM images and (c) EDAX spectrum of Mn 2 O 3 NPs.

Figure 4 :
Figure 4: Antibacterial activity of PF-127-coated Mn 2 O 3 NPs.NPs inhibit the growth of bacteria and fungi.Antibacterial activity was determined for PF-127-coated Mn 2 O 3 NPs by measuring the zone of inhibition.

Figure 6 :
Figure 6: Apoptosis was determined in MDA-MB-231 and MCF-7 cells by using AO/EtBr dual staining with IC 25 and IC 50 doses of PF-127-coated Mn 2 O 3 NPs after 24 h of treatment.MDA-MB-231: control cells (a), cells treated with IC 25 (b), and IC 50 (c) of PF-127-coated Mn 2 O 3 NPs.MCF-7: control cells (d), cells treated with IC 25 (e), and IC 50 (f) of PF-127-coated Mn 2 O 3 NPs.This is a representative image of the experiment performed in triplicate at a magnification of 20×.

Figure 7 :
Figure 7: DAPI nuclear staining was used to determine apoptosis in MDA-MB-231 and MCF-7 cells.MDA-MB-231: control cells (a), cells treated with IC 25 (b), and IC 50 (c) of PF-127-coated Mn 2 O 3 NPs; MCF-7: control cells (d), cells treated with IC 25 (e), and IC 50 (f) of PF-127-coated Mn 2 O 3 NPs.This is a representative image of the experiment performed in triplicate at a magnification of 20×.

Figure 8 :
Figure 8: In MDA-MB-231 and MCF-7 cells treated for 24 h with PF-127-coated Mn 2 O 3 NPs, the mitochondrial membrane potential was determined by JC-1 staining.MDA-MB-231: control cells (a), cells treated with IC 25 (b), and IC 50 (c); MCF-7: control cells (d), cells treated with IC 25 (e), and IC 50 (f).This is a representative image of the experiment performed in triplicate at a magnification of 20×.

Figure 9 :
Figure 9: A fluorescence microscope image stained with DCF-DA.MDA-MB-231: control cells (a), cells treated with IC 25 (b), and IC 50 (c) of PF-127-coated Mn 2 O 3 NPs; MCF-7: control cells (d), cells treated with IC 25 (e), and IC 50 (f) of PF-127-coated Mn 2 O 3 NPs.This is a representative image of the experiment performed in triplicate at a magnification of 20×.

Figure 10 :
Figure 10: Annexin-V/-FITC/PI Flow cytometry analysis of MDA-MB-231 and MCF-7 cancer cells treated with IC 50 concentration of PF-127-coated Mn 2 O 3 NPs for 24 h.These figures are from representative experiments carried out in at least two independent tests.MDA-MB-231: control cells (a), cells treated with IC 25 (b), and IC 50 (c); MCF-7: control cells (d) cells treated with IC 25 (e), and IC 50 (f).Data were mean ± SD of two independent experiments.*p < 0.05 when compared with control, **p < 0.001 when compared with control.

Figure 11 :
Figure 11: Cell cycle analysis using flow cytometry after staining with PI.MDA-MB-231 and MCF-7 cells were treated with IC 25 and IC 50 concentrations of PF-127-coated Mn 2 O 3 NPs for 24 h.Cell cycle pattern and apoptosis distribution in MDA-MB-231: control cells (a), treated with IC 25 (b) and IC 50 (c), and percentage of cells distribution (d); MCF-7: control cells (e), treated with IC 25 (f) and IC 50 (g), and percentage of cells distribution (h).
[30]ct of NPs synthesized using the GU leaf extract on breast cancer  3 and Escherichia coli) and Candida albicans using the disc diffusion technique suggested by Elderdery et al.[30].