Zinc oxide-manganese oxide/carboxymethyl cellulose-folic acid-sesamol hybrid nanomaterials: A molecularly targeted strategy for advanced triple-negative breast cancer therapy

: Multifunctional nanocomposites (NC) can greatly enhance therapy outcomes by reducing tumor proliferative potential. We created a novel class of Zn_Mn_CMC_FA_sesamol NC in the current work to combat breast cancer (MDA-MB-231) cells. To understand how zinc (Zn), manganese (Mn), carbox-ymethylcellulose, and folic acid (FA) interact with sesamol, UV-Visible spectrophotometer and Fourier Transform Infrared spectroscopy were used to analyze the absorption behavior of the synthesized NC. The particle size of NC was con ﬁ rmed by X-ray di ﬀ raction and dynamic light scattering. Scanning electron microscopy was used to assess the morphological features of these NCs. photoluminescence spectrum was used to analyze the optical and electron transition molecules of the sample. In addition to MTT analysis, acridine orange/ethidium bromide (AO/EtBr) analysis of reactive oxygen species (ROS) and nuclear staining with 4 ′ ,6-diamidino-2-phenylindole as well as ﬂ ow cytometry were used to con ﬁ rm the apoptotic activity of Zn_Mn_CMC_FA_sesamol NC on MDA-MB-231 cells. The results showed signi ﬁ cant cytotoxicity, apoptosis induction on AO/EtBr, and increased ROS production in treated cells compared to control cells. The cell cycle analysis revealed that NCs triggered apoptosis and arrested the cell cycle in G0/G1 phases. As a conclusion, the created NC serves as a versatile platform for the successful molecularly targeted


Introduction
Breast cancer is the most prevalent type of cancer in women and the leading cause of death from cancer in women worldwide.In 2018, there were more than 2.05 million new cases found and the incidence is projected to rise by more than 46% by 2040, according to GLOBOCAN [1].Depending on the cell of origin, breast cancer can vary in form and severity.Connective tissue, lobules, and ducts make up the three primary structural components of the breast.The lobes and ducts are where the majority of breast cancers begin [2].Breast cancer officially overtook lung cancer as the most common cancer in the world on December 15, 2020, according to statistics on the global burden of cancer published on the official website of the World Health Organization's International Agency for Research on Cancer [3].They also stated that 2.26 million new cases of breast cancer had been diagnosed globally.Although individuals with breast cancer have a higher survival rate, it is hard to ignore the disease's high incidence, earlier onset, high metastasis, and bad prognosis [4].Several types of breast cancer cell lines are investigated for anti-cancer activity, some examples of cell lines are MDA-MB-468, MDA-MB-231, MCF-7, and BT-474, etc., One of the most popular triple-negative cell lines for metastatic breast cancer research is MDA-MB-231 [5].It accounts for 15-20% of all cases of breast cancer and possesses low levels of HER-2 expression, no progesterone receptors, and no estrogen receptors, making it challenging to treat with medication in targeted therapy [6].As a result of poor prognosis, tumors can easily spread to internal organs in 1-3 years and 40% of them spread to the lungs.Since Triple-negative Breast Cancer (TNBC) has a poorer prognosis than the other subtypes of breast cancer, it is essential to identify new biomarkers and design efficient treatment strategies [7].
Surgery, chemotherapy, radiation, immunotherapy, and hormone treatment are all part of the current standard of care for breast cancer.All of these have the potential to cause adverse effects and may not entirely eradicate the tumor [8].Novel medications are still needed, especially for patients with breast cancer that has been inadequately treated.New perspectives for cancer therapy have emerged as a result of the development of nanomedicines.The prevalence of breast cancer has been linked in several studies to food, specifically dietary patterns.To enhance targeted delivery methods and lessen their toxicity to healthy cells, nanoparticles are utilized in anti-cancer therapy [9,10].Metal nanoparticles have recently attracted a lot of scientific attention and have emerged as the most promising field of study due to their unique characteristics [11].For instance, it has been demonstrated that dietary antioxidants control oxidative stress in the body and that a larger consumption of antioxidants is linked to a decreased risk of breast cancer [12].Nanocomposites (NCs) have RNA interference.RNA interference (RNAi) therapy based on nanoparticles delivers the siRNA molecule(s) for gene silencing, which may be used as a cancer treatment.RNAi molecules can be delivered to cancer cells using polymeric nanoparticles, where they can impede the expression of genes linked to the development and spread of tumors [13].
The clinical efficiency of nanoparticle treatments has been demonstrated by the production of nanoparticle drug conjugates in various stages of clinical therapy.Several papers have reported on the production of the metalorganic framework and carboxymethyl cellulose (CMC) NCs, respectively [14,15].An anionic water-soluble biopolymer is CMC, one of the cellulose derivatives.The unique qualities of CMC include hydrophilicity, biodegradability, nontoxicity, pH sensitivity, and biocompatibility.It is utilized as a carrier that has generated a lot of attention in biological applications because of these CMC properties [16][17][18].Folic acid (FA) is crucial for cell maintenance and proliferation, and its receptors are overexpressed on tumor cells in various cancers.This upregulation suggests that FArelated therapeutic agents may have reduced toxicity and enhanced potency against tumor cells.FA and FA conjugates bind to folate receptors with high affinity, and FA-modified drug delivery vectors can transfer therapeutic agents to tumor cells with amplified folate receptor expression [19][20][21].
Sesame oil stands out among other vegetable oils because of its wide range of applications and excellent therapeutic potential.The acylglycerols (oleic acid, linoleic acid, palmitic acid, stearic acid, and arachidic acid) and other sesame lignans, such as sesamin, sesamol, and sesamolin provide the distinctive potential to sesamol oil.Sesamol has a strong antioxidant activity, which protects membranes from lipid peroxidation [22].Sesaminol enhances the availability of vitamin E from tocopherols and inhibits membrane lipid peroxidation by increasing liver and plasma concentrations of tocopherols [23].Sesaminol has a modest therapeutic benefit against breast cancer, nevertheless.As a result, we sought to create an elaborate action by integrating sesamol with zinc, FA, and manganese(II) nitrate hexahydrate, and CMC, which would soon open a new door.Sesaminol's availability in nature, structural chemistry, and the creation of nanocarriers to boost bioavailability are its main advantages.As a result, the goal of this work was to reveal the molecular basis of the synthesized NCs' effects on MDA-MB-231 cells, to gather the data required to start successfully developing a new anticancer drug from sesaminol.
2 Materials and methods 2.1 Preparation of sesamol NCs 0.2 g ZnO and 0.1 M manganese(II) nitrate hexahydrate (Mn (NO 3 ) 2 •6H 2 O) are added to 90 mL of distilled water together with 500 mg dissolved CMC, 100 mg FA and 50 mg sesamol to create a mixed solution.Then, 0.1 M NaOH was added gradually and the mixture was heated with magnetic stirring for 6 h at a temperature of around 60°C.The resulting nanopowder was washed with the mixture of ethanol and deionized water at different time points until the required pH values were reached.The precipitate was allowed to dry at 120°C for an hour to produce ZnO_Mn_CMC_FA_sesamol NCs and the nanopowder was subsequently annealed at 200°C for 2 h [24].All the chemicals used in this study were obtained from Sigma-Aldrich Company (Massachusetts, United States).

Characterization studies
UV-Visible spectrophotometer (UV-VIS) and photoluminescence spectroscopy (PLS) were used to examine the optical characteristics of the NC synthesized.X-ray diffraction (XRD) and dynamic light scattering (DLS) were used to determine its physical properties and particle size.VEGA3TESCAN Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) were also used to identify surface morphology.The presence of functional groups in the synthesized nanoparticles was confirmed using Fourier Transform Infrared (FT-IR) spectroscopy [25].

Anticancer activity 2.3.1 Cell culture
Breast epithelial cell line (HBL-100) and TNBC cell line (MDAMB-231), which are estrogen-negative, were both grown in 10% fetal bovine serum, supplemented with a minimum essential medium, at 37°C in a humid incubator with 5% carbon dioxide.When the cells from the T75 flasks were sub-confluent, they were trypsinized and seeded in 96-well plates.

Viability assessment
The cells were grown for 24 h with a seeding density of 1 × 10 4 cells per well.Sesamol NCs are added at concentrations ranging from 3.13 to 200 µg•mL −1 .After 24 h, the cytotoxic impact on monolayer cells was evaluated.The cells were stained using 200 µL•well −1 of MTT solution in phosphate-buffered saline (PBS) (0.5 mg•mL −1 ) for 4 h at 37°C.After removing the media, 100 µL of dimethyl sulfoxide was added to each well to dissolve the MTT formazan crystals.An ELISA microplate reader was used to measure the absorbance at 492 nm.Each experiment was performed three times.Finally, the relative cell viability dose-response curve was used to calculate IC50 values or the quantity of chemicals that caused 50% of cell death [26].

Apoptotic cell death in the breast cancer MDA-MB-231 cells
Apoptosis induced by sesamol NCs and paclitaxel in MDA-MB-231 was detected using dual stain acridine orange and ethidium bromide (AO-EtBr).The AO-EtBr stain was prepared by mixing at a 1:1 ratio.Sesamol NCs were administered to cancer cells for 24 h after 80% growth based on the IC50 for each cell type.After removing the medium, AO-EtBr stains at 50 µL•well −1 were added.After 20 s, the stain was removed and apoptotic cells were analyzed using an inverted fluorescent microscope.ImageJ (Olympus Corporation, Beijing, China) was used to compute the results, which allowed for the separation of viable cells from apoptotic ones [27].

Nuclei morphological change assay
By using a 4′,6-diamidino-2-phenylindole (DAPI) test, the impact of sesamol NCs on MDA-MB-231 cell nucleus morphological alterations was discovered.The cells were plated in 24-well dishes and allowed to grow for 24 h.The cells were subsequently given a 24 h treatment with paclitaxel and sesamol NCs.After washing with PBS, cells were fixed with 100% methanol and stained using 1 mg•mL −1 DAPI solution for 30 min at 37°C.To stain the cells, wash PBS after surplus dye was removed.Under an inverted fluorescent microscope (Olympus Corporation, Beijing, China), the morphological alteration of the nuclei was seen [28].

Reactive oxygen species (ROS) activity assay
The MDA-MB-231 cell line was subjected to various dosages of sesamol NCs, and the formation of ROS in the cell line was examined using a fluorescence microscope.After being exposed to sesamol dosages for 24 h, the cells were then treated with 10 mM of DCFH-DA for 30 min at 37°C.Each well received 200 mL of PBS instead of the reaction mixture, which had been aspirated and observed under an inverted fluorescent microscope.The percentage of fluorescence intensity compared to the control wells was used to represent the values [29].

Flow cytometry for apoptosis
To analyze the cell cycle phases, we collected the cells, processed them with 70% ethanol, and then incubated them for 12 h.Later, the cells were rinsed using saline, and then 300 µL of staining solution consisting of propidium iodide (PI; 100 µL), proteinase inhibitor (0.08 mg•mL −1 ), and RNase (0.5 mg•mL −1 ) was added to the cells for 30 min.
The DNA-associated PI fluorescence was assessed using flow cytometry, and the proportions of cell nuclei in various cell cycle stages, such as G1, S, and G2/M, were examined using the MultiCycle software (Phoenix Flow Systems, San Diego, USA) [30].

Statistical analysis
The values are analyzed statistically using SPSS software with one-way ANOVA and Tukey's post hoc assay.The data are expressed as mean values ± SD of triplicates, with p < 0.05 indicating significance.

DLS, PL spectrum, and FT-IR
The size distribution of the NC was validated by DLS measurement, with the dispersion ranging from 10 to 100 nm.The average size of the sesamol NCs synthesized was 191.00 nm (Figure 2a).The PL spectra revealed an emission peak at 438.77 nm, which corresponds to photons, and shoulder peaks at 482.18 nm, which corresponds to electron transition, and 515.26 nm, which relates to oxygen vacancy (Figure 2b).FT-IR spectra confirmed the presence of specific bands for several functional groups of synthesized NC (Figure 2c).Due to the fingerprint region of nanoparticles at the peak of 400-600 cm −1 in the figure, the Peak at 674-579 cm −1 indicates the presence of ZnMnO.The broad peak 3,436 cm −1 is assigned to the OH stretching vibration of the phenolic group.The peak observed at 2,924.3 cm −1 is due to the C-H stretching frequency of the alkane group.1,627 cm −1 peaks are assigned to the NH stretching vibration of the -NH2 group.The peaks found at 1,440 and 1,271 cm −1 were assigned to the stretching vibration of the (COO-) carboxyl group.862 and 955 cm −1 are responsible for the C-O stretching vibration of the methylenedioxyphenol group.These FT-IR results suggest the presence of functional groups in the sesamol NC.

SEM and TEM
SEM and TEM examinations were used to explore the structural characteristics of ZMCFA sesamol NCs.The particle shape of ZnMnO NC is seen in Figure 3.The SEM picture displays cubic and spherical aggregate forms.The TEM images of the ZnMnO NC are shown in Figure 4. Crystalline grain has approximately spherical and cubic forms in its morphology.

Energy dispersive X-ray (EDAX) spectroscopy
The EDAX spectroscopy (Figure 5) determined the constituent elements of the NC.This analysis also provided   Zn_Mn_CMC_FA_sesamol NCs targeted approach for breast cancer  5 information on the weight percent of ZnMnO, which revealed a 14.20 wt% of zinc, 0.78 wt% of manganese, and a 40.30wt% of oxygen composition.

3.2
In vitro study

Cytotoxicity study
For both types of cells, the observed percentage of cell viability was discovered to be dosage-dependent up to 24 h (Figure 6).ZMCFA sesamol NCs only had a dose-dependent cytotoxic impact on MDA-MB-231 and HBL-100, with an IC50 of 18.3 µg•mL −1 .Additionally, the increased concentration of functionalized NC's showed no harmful effects on HBL-100 even at greater concentrations ranging from 3.13 to 200 µg•mL −1 , demonstrating the material's biocompatibility and gradual and steady drug release capabilities.

Apoptosis induction by ZMCFA sesamol NCs
One of the most used fluorescent dyes, AO/EtBr, when used to stain treated cells revealed the morphological characteristics of necrotic cells and cells going through various stages of apoptosis.The cytoplasm and nucleus of living cells were fluorescently marked in green.EtBr was accumulated in apoptotic cells, resulting in condensed and shattered nuclei.The nuclear structure of necrotic cells was identical to that of live cells, they were orange in color, and they lacked condensed chromatin.The findings showed that paclitaxel (25 nM) and ZMCFA sesamol NCs (IC50 of 18.3 µg•mL −1 ) therapy greatly increased breast cancer cell death and apoptosis induction compared to the control group (Figure 7).

Nuclei morphological changes using DAPI by ZMCFA sesamol NCs
Using the DNA-specific fluorescent probe DAPI to label the nuclei allows researchers to see the morphological changes that occur during apoptosis in breast cancer cells (Figure 8).As a result, cells treated with sesamol NC (IC50 of 18.3 µg•mL −1 ) have chromatin-filled nuclei that have gathered and are fully fractured, which are signs of apoptosis.The cellular nuclei in the control group are rounded   In MDA-MB-231, the IC50 of the nanocomposite is 18.3 µg/mL, while the HBL-100 is infinite.
Zn_Mn_CMC_FA_sesamol NCs targeted approach for breast cancer  7 and uniformly stained with DAPI.Similar to paclitaxeltreated cells (a positive control), the cells exposed to sesamol NCs exhibited chromatin aggregates and apoptotic bodies.Cells treated with paclitaxel exhibit well-distributed apoptotic bodies, a hallmark of apoptosis.

Generation of intracellular ROS by sesamol NC
The amount of ROS in cells labeled with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was measured in this investigation to determine if treating MDA-MB231 cells with ZMCFA sesamol NCs may promote ROS accumulation.As a result, ZMCFA sesamol NCs may cause more DNA damage, mitochondrial instability, and eventually death owing to intracellular ROS production as shown in Figure 9.In treated MDA-MB-231 cells, the DCFH-DA stain's fluorescence intensity was visible notably, and concentration IC50 of 18.3 µg•mL −1 of sesamol NC promoted ROS production during the course of a 24-h treatment period.

Cell cycle analysis with flow cytometry and PI by sesamol NCs
To determine if the anti-proliferative impact was caused by cell cycle arrest, flow cytometric measurement was done after a 24-h treatment with sesamol NC and paclitaxel.However, following exposure to sesamol NC at18.3 µg•mL −1 , compared to the paclitaxel treated and control group after 24 h, substantial modifications were produced in the cell cycle (G0/G1 phase) (Figure 10).Interestingly, after 48 h of exposure to all doses, the build-up of cells began in the sub-G1 phase.With exposure to sesamol NC 18.3 µg•mL −1 at 12 and 48 h, DNA accumulation was seen in the G0/G1 phase, and the number of cells in the S phase was significantly reduced.

Discussion
We investigated the sesamol NC as an anti-breast cancer medication.The absorbance of the sample is affected by numerous parameters, including band gap, oxygen deficit, surface roughness, and impurity.The UV-vis absorption spectra of NC exhibited maximal absorption at 285 and 357 nm (Figure 1a).FA's UV absorbance was measured at  280 nm [31].The absorption spectrum shifted to 285 nm, which encompasses sesamol and FA UV absorbance.
Because the spectrum featured the typical UV absorbance peaks of encapsulated sesamol, FA, and ZnMnO, the absorbance spectrum at 357 nm validated the encapsulation of the sesamol conjugation of FA and coordination of ZnMnO. Figure 1b where k is a constant (9.4), and only D is a constant.D stands for peak width at half-maximum along the (101) plane and Bragg's diffraction angle, and D is the wavelength (1.5406 for CuK).The particle size is 52 nm on average.The hydrodynamic diameter of the sesamol NC was evaluated using DLS to get particle size information (Figure 2a).The composite was 191 nm in size, since the DLS particle size was larger than the XRD values and the NCs were submerged in water.This is known as hydrodynamic size.
The PL spectroscopies of a synthesized NC generated at 350 nm are shown in Figure 2b.Emissions were detected at 488, 482, and 515 nm.The peak at 488 nm (near the band edge) is due to radiative recombination caused by the free exciton-exciton collision mechanism.The peak at 515 nm represents oxygen vacancies (Ov).The intense luminescence at 482 nm was caused by electronic transitions inside the d levels of the Mn 2+ metal ion.This might be due to the coordinate link established between the FA-CMC-OH groups and the metal ions Zn and Mn.The FT-IR spectroscopy of the sesamol NC is shown in Figure 2c, respectively.The presence of phenolic OH groups is confirmed by the broad peak at 3,436 cm −1 , and it indicates that the sesamol compounds are encased by the nanocomposite.Furthermore, the presence of FA in the NC is confirmed by the (COO-) carboxyl group peak locations at 1,440 and 1,271, and the NH2 stretching vibration peaks at 1,627 cm −1 .The absorbance peaks 579, 619, and 674 cm −1 are responsible for the stretching frequency of the ZnMnO NC. Figure 3 shows that ZnMnO CMC FA and sesamol contain synthetic NC, and that ZnMnO NC has a cubic and spherical shape.The structural characterization of ZnMnO NC has a cubic and spherical shape, as seen in Figure 3.The SEM picture reveals that the NC aggregates severely, which is likely due to the high surface energy of ZnO nanoparticles [33].The TEM was used on the samples to better understand the morphology of the NC.The TEM images of ZnMnO nanoparticles are shown in Figure 4.The crystalline grain form is approximately spherical and cubic, which is compatible with the wurtzite structure found by the XRD result.The HR-TEM picture of the NCs displays the lattice fringes of the produced components.
We examined the proliferation effects of ZMCFA sesamol NCs on breast cancer cells and found that it had no adverse effects on either the MDA-MB-23 or the HBL-100 cell lines, suggesting that it could be useful as a non-toxic drug carrier for cells.This may be because Zn, Fe, and Mg are important micronutrients that are involved in all major metabolic pathways.However, at higher concentrations, they may interfere with several cellular processes, such as catalytic, structural, and regulatory functions, which would inhibit cell proliferation.These sesamol NCs have a specific anti-proliferative effect on breast cancer cells as a result of the sesame bioactive component; it demonstrated a greater selectivity against breast cancer cells.Similar to this, sesamol NCs reduced the vitality of cancer cells and prevented the colony formation of HepG2 cells [34].Also, sesamin NCs had the same anti-proliferation action against MDA-MB-231 at an IC50 value of 51.1 µmol•L −1 [35].Sesamol NC's cytotoxic effects and apoptosis seemed to cause less cell viability and more apoptosis compared to control cells.In cancerous cells, sesamol causes growth arrest and apoptosis.However, due to inadequate bioavailability, its medicinal importance is constrained.Although sesamol NCs contain phenolic chemicals that cause cancer cells to undergo apoptosis and a growth arrest [36].It has been discovered that sesame oil can inhibit the start of apoptosis and the production of proteins that are protective against HFD-induced ER stress.According to our research, the ZMCFA sesamol NCs were highly effective against the MDA-MB-231.As a result, the usage of ZMCFA sesamol NCs proved as a more effective treatment.Sesamol NC's cytotoxic actions cause mitochondrial integrity to become unstable and caspases to get activated, which results in cell death (apoptosis).The cytoplasm of cells treated with NC fluorescence uniformly, and a few cells also showed a specific halo surrounding the nucleus, which is likely located inside the nuclear membrane.With no visible fluorescence in the nuclear membrane or nucleus, cells treated with paclitaxel revealed a nuclear-centric pattern of localization.Sesamoltreated cells exhibited chromatin aggregates and apoptotic bodies similar to paclitaxel-treated cells [37].This study showed that sesamol increased apoptosis leading to DNA disruption, which caused mitochondrial dysfunction, and well-separated apoptotic bodies were found in cisplatin-treated cells.A distinct sign of apoptosis, well-separated apoptotic bodies, were seen in cisplatin-treated cells.
The ZMCFA sesamol NCs would synergistically increase ROS production.They contend that medications delivered via ZMCFA sesamol NCs cause DNA damage, ROS, and death in tumor cells.Therefore, it was determined that ZMCFA sesamol NCs were potential materials to promote ROS generation which activates intrinsic apoptotic proteins in cancer cells.As a result, the control of cell apoptosis depends critically on the creation of elevated amounts of ROS [38].The release of apoptotic cells, which increases the cytotoxic impact, is triggered by the destabilization of the mitochondrial membrane and activation of signal molecules, both of which are caused by ROS.The development of SNEDDS (self nanoemulsifying drug delivery systems) has a significant effect on MCF-7 cells without harming healthy cells, as evidenced by the increase in ROS generation and DNA fragmentation [39].
The modulation of the cell cycle is an important strategy in the development of anticancer medicines [40].The G1 phase is characterized by cell growth, RNA production, and protein synthesis for DNA formation.In typical circumstances, DNA replication and cell growth occur during the S phase, whereas new protein synthesis occurs during the G2 phase.Nuclear and cytoplasmic divisions occur at the M developmental stage [41].A reduction in the percentage of G1 and S phase cells and the observation of cell aggregation in the sub-G1 phase after 48 h of treatment demonstrate that all doses of ZMCFA sesamol NCs induced apoptosis.Similarly, sesamin exerted cytostatic effects at 100 µM and inhibited cell development by arresting it in the G1 phase [42].In another study, sesamol arrested the cell cycle at the S phase leading to DNA damage and inhibition of DNA replication and an increased ratio of G0/G1 in HepG2 cells confirming the initiation of apoptosis [34].Hence, ZMCFA sesamol NCs may be used as potential anticancer agents due to these intriguing prospective properties.

Conclusion
The goal of the work was to create multifunctional NC materials that could be employed for both imaging and treating TNBC.The cytotoxic effects of the NC on breast cancer cells were in a dose-and time-dependent manner with no discernible effects on healthy cells.ROS and apoptotic bodies were released to internalize NC-induced mitochondrial apoptosis.The NC induced apoptosis by intercalatively binding with DNA, stopping the cell cycle, and destroying DNA.The study we conducted, to our knowledge, is the first to demonstrate that synthesized NC has the potential to be employed in vitro as an effective and secure anti-cancer drug.The NC from the study can be made using more environmentally friendly methods and is safe and biocompatible.Further investigation into the mechanism of NC that is specific to cancer cells and the development of NC-based nanosystems containing drugs and antibodies will aid in the development of potential nanostructures for imaging and therapy against cancer models.As a result of our research, functionalized NC will become a well-known, adaptable platform for drug delivery that may effectively target breast cancer.As a result, it holds the key to realizing the potential of breast cancer treatment.The results showed that ZMCFA_sesamol NCs kill MDA-MB-231 cell lines by reducing cell growth, antioxidant state, and apoptosis.Additionally, assessing its stability and release profile in physiological conditions would provide valuable information for its potential use in medical applications.

Figure 6 :
Figure 6: ZMCFA sesamol NCs cause cytotoxicity in MDA-MB-231 cells.MDA-MB-231 cell line was exposed to ZMCFA sesamol NCs at several doses (3.13-200 µg/mL) for 24 h.The MTT test was performed on the cells, and the results are shown as the mean standard deviation of three separate trials.In MDA-MB-231, the IC50 of the nanocomposite is 18.3 µg/mL, while the HBL-100 is infinite.

Figure 7 :
Figure 7: Effect of ZMCFA sesamol NCs on the apoptotic cell death in the breast cancer MDA-MB-231 cells.The apoptosis-free green fluorescence of the control cells suggests that they are still alive.The ZMCFA sesamol NCs treated cells displayed yellow and orange fluorescence, respectively, signifying early and late stages of apoptosis and necrotic cells with condensed or shattered nuclei.Red cells indicated necrotic cell death.Similar to paclitaxel-treated cells (positive control).This is a representative image of the experiment performed in triplicate with magnification at 20× and Scalebar = 50 µm.

Figure 8 :
Figure 8: Fluorescence microscope image of nuclear damage induced by ZMCFA sesamol NCs stained with DAPI.DAPI nuclear condensation test was followed by 24 h treatment with the ZMCFA sesamol NCs.The cellular nuclei in the control group are rounded and uniformly stained with DAPI.Similar to paclitaxel-treated cells (a positive control), the cells exposed to sesamol nanocomposites exhibited chromatin aggregates and apoptotic bodies.This is a representative image of the experiment performed in triplicate with magnification at 20× and Scalebar = 50 µm.

Figure 9 :
Figure 9: Intracellular ROS generation induced by ZMCFA sesamol NCs stained with DCF-DA.The control cells revealed poorly fluoresced cells, contrastingly the ZMCFA sesamol NC treated MDA-MB-231 cells for 24 h demonstrated higher fluorescence, which unveils higher endogenous ROS accumulation in the MDA-MB-231 cells.Similar to paclitaxel-treated cells (a positive control), the cells exposed to sesamol nanocomposites exhibited ROS accumulation and apoptotic bodies.This is a representative image of the experiment performed in triplicate with magnification at 20× and Scalebar = 50 µm.

Figure 10 :
Figure 10: Cell cycle analysis using flow cytometry after staining with PI.MDA-MB-231 cells were treated for 24 h with ZMCFA sesamol NCs at its IC50 of 18.3 µg/mL concentration and with paclitaxel at a concentration of 25 nM.Cell cycle pattern in control cells (a), cells treated with ZMCFA sesamol NCs (b), cells treated with paclitaxel, a positive control (c), and the proportion of cells in each distribution of cycles (d).(* denotes a p-value less than 0.05 compared to the control; **, a p-value less than 0.001).