Materials ferric chloride hexahydrate (≥99% purity), ferrous sulfate heptahydrate (≥99% purity), hydrochloric acid (37%, ACS reagent), sodium hydroxide (≥97.0%, pellets), ammonium hydroxide (≥28% purity), Morin hydrate (≥100% purity), methanol (≥99.8%, ACS reagent), dimethyl sulfoxide (DMSO) (≥99.9%, ACS reagent), ethanol (96%, ACS reagent)and zinc chloride (≥98% purity) were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade. Double-distilled water was used for synthesis, solution preparation, and other purposes throughout the experiment.

The leaves of the okra plant were first washed with tap water several times and then washed and cleaned with deionized water 5 times to remove dust and contaminants adhering to the surface of the leaves. After the cleaning process, drying was done in the open air in a fume hood at room temperature, and the dried okra leaves were ground into a powder with the help of an IKA M20 Universal grinder and stored in a container to be used in OLAC production. 10 g of the powder sample obtained by grinding the leaves of the okra plant after drying were weighed, taken into a 500 mL erlenmayer, then, a solution of zinc chloride with a concentration of 1 M (for activation of activated carbon) was added and mixed in a shaking water bath at 80°C for 1 hour (Veerakumar et al., 2016). The precipitate of the mixture, which was brought to room temperature and cooled, was placed in a glass crystallization container to be homogeneous and dried in an oven at 100°C for 24 h. Afterward, the dried mixture was placed in porcelain crucibles and subjected to carbonization in the muffle furnace set at 600°C. The OLAC obtained after the carbonization process was cooled in a desiccator at 25°C. Washing was carried out several times with 0.1 N HCl to remove unreacted zinc, chlorine, and other ions on the surface of OLAC. After washing with HCl, the product was washed with deionized water (until the acidity was removed and the pH value became neutral) and left to dry in an oven at 70°C for 24 h (Farma et al., 2023; Komal et al., 2024).

As reported in the literature, with some modifications, the surface of MNPs was synthesized by coating them with AC obtained from the okra plant (Inbaraj et al., 2021). 3.0 g of iron (III) chloride hexahydrate was dissolved in 50 mL of deionized water in an argon gas atmosphere. In the next step, 2.1 g of iron (II) sulfate heptahydrate was added to the solution containing Fe³⁺ ions and the temperature of the mixture was slowly increased to 90 °C in the presence of argon gas. After 30 min, 10 mL of 26% ammonium hydroxide solution was added to the mixture, and a black solution was formed. This resulting solution continued to be mixed in the magnetic stirrer for another 30 min under the same conditions. Then, 500 mg of OLAC solution dissolved in 100 mL of water was added to the mixture. After the addition of the solution containing OLAC, the product in the magnetic stirrer was mixed for 60 min and the resulting MNPs@OLAC nanocomposite was allowed to cool at room temperature, and the MNPs@OLAC nanocomposite was separated from the solution medium using a magnet. The MNPs@OLAC nanocomposite was washed several times with deionized water and dried in a lyophilizer. After the drying process, the MNPs@OLAC nanocomposite was stored in a colored container to be used in experimental studies.

For the purpose of to loading MH into the MNPs@OLAC magnetic nanocomposite, 10 mL of a 100 μg/mL MH solution dissolved in methanol was added to 50 mg of the nanocomposite. The mixture was then stirred in a water bath at room temperature for 24 hoursThen, the particles were isolated from the solution using an external magnet based on their magnetic properties, and the MH concentration in the solution was measured at a wavelength of 385 nm using UV-Vis spectrophotometry. To calculate drug loading, it was obtained by subtracting the MH concentration in the supernatant from the initial MH concentration using Equation 1. Subsequently, magnetic nanoparticles which contained the medication were isolated by the application of a magnetic field and thereafter underwent a drying procedure. Drug loading efficiency  % = Total   amount   of   drug − Free   drug   in   the   supernatant   Total   amount   of   drug × 100 (1)

For the purpose of the MH release investigation, 10 mg of dry drug-loaded nanoparticles were analyzed in 5 mL of PBS with a pH of 5.4 and 7.4 at a temperature of 37°C while being stirred for durations of 1, 3, 6, 12, 18, 24, 36, 48, 60, and 72 h. UV-Vis spectrophotometry was performed at a wavelength of 385 nm to determine the amount of medication that was released after the set incubation periods had passed During the course of the drug release tests, measurements were carried out in accordance with Equation 2. Drug release  % = Released   drug Total   drug × 100 (2)

An examination of the viability of the cells was conducted out at the Cell Culture Laboratory of the Dicle University Faculty of Veterinary Medicine Laboratory. The healthy cell line (HUVEC) and the cancer cell lines (MCF-7, U373, T98-G, and HT-29) were obtained from the “American Type Culture Collection” (ATCC).

In accordance with the findings of Baran, the chosen cell lines were grown in a cell culture medium contained within a T75 flask and then incubated at 37°C in an atmosphere containing 5% carbon dioxide (Baran, 2023). After the cells had attained a confluency level of 80%–90%, they were removed from the flasks and the hemocytometric method was used to determine the total number of cells. To carry out two separate time applications, 24 and 48 h, the cells whose numbers were calculated were injected onto 96-well plates in sizes of 10 × 10⁵ in three replicates, with 90 µL of media in each well. Additionally, the cells were inoculated into microplates in triplicate for MNPs@OLAC-MH in pH 7.4 and MNPs@OLAC-MH in pH 5.4 with the intention of doing two separate time applications. The cells were waited for 24 h to adhere to the microplate bottom. The next day, MNPs@OLAC-MH at pH 7.4 and MNPs@OLAC-MH at pH 5.4 were applied to the seeded plates at various concentrations (1, 50, 100, and 200 μg/mL). Liquid with pH 7.4 for MNPs@OLAC-MH and pH 5.4 for MNPs@OLAC-MH was applied to the cells in the control group.

MTT test was conducted at 24 and 48 h following the application, to assess alterations in cell viability. Each well-containing cells in the microplate was supplemented with 10 µL of the prepared MTT solution (5 mg/mL) and then incubated for 3 h at 37°C in a humid atmosphere with 5% CO₂. Following a 3-h duration, the medium was removed and 100 μL of DMSO were added to each well. After a 20-min incubation period on the shaker, the optical density (OD) values of the wells were measured using UV/Vis Spectrophotometry (Schneider et al., 2021; Montazersaheb et al., 2024).

The average absorbance values obtained by reading the control wells were accepted as 100% live cell values. The absorbance values obtained from the MNPs@OLAC-MH in pH 7.4 and MNPs@OLAC-MH in pH 5.4 applied wells were accepted as % viability by proportioning them to the control absorbance value. MTT trials were repeated three times on different days.

The data collected from the study were analyzed using the IBM SPSS 21.0 software package. The statistical significance level was deemed acceptable at a p-value of less than 0.05. Data were compared between groups using unpaired t-tests and among multiple groups by one-way ANOVA, followed by Tukey’s post hoc tests. P < 0.05 was considered statistically significant.

Figure 1 shows the characterization results of MNPs@OLAC and MNPs@OLAC-MH nanocomposites by FTIR spectroscopy. The FT-IR spectrum of MNPs@OLAC nanocomposite at 3365 cm⁻¹ is attributed to the stretching and vibration of the–OH (alcoholic and phenolic) group (Ma et al., 2022). When the spectra of MNPs@OLAC and MNPs@OLAC-MH are examined, the peak at 2109 cm⁻¹ corresponds to the C≡C stretching of alkynes (Emenike et al., 2023), when the spectrum of MNPs@OLAC and MNPs@OLAC-MH nanocomposites at 2113 cm⁻¹ is examined, the measured peak is attributed to the presence of C-H and alkyne groups (Xavier, 2021; Mazumdar et al., 2015; Ferreira et al., 2017) and the peaks observed at 1990–1994 cm⁻¹ indicate stretching vibration of C=C bonds (in alkynes) (Al and Kabakcı, 2024). The peaks seen at 1602 and 1636 cm⁻¹ (C=O group) correspond to the presence of hydroxyl and carboxylic acid groups (Ezeonuegbu et al., 2021). In addition, for MNPs@OLAC and MNPs@OLAC@MH nanocomposites, the absorption of the peaks at 1039 and 1080 cm⁻¹ indicates the presence of C-O and C-N groups (He et al., 2022; Sun et al., 2020). When the spectra of MNPs@OLAC and MNPs@OLAC@MH are examined in Figure 2, the peak seen at 533 and 536 cm⁻¹ indicates the presence of Fe-O bond (Tural et al., 2024).

The surface images of MNPs@OLAC and MNPs@OLAC-MH nanocomposites were analyzed using SEM. SEM images of MNPs@OLAC and MNPs@OLAC-MH nanocomposites are shown in Figures 2A, B. The MNPs@OLAC nanocomposite has different pore diameters and a granular surface. The reason for the smoothness on the surface of the MNPs@OLAC nanocomposite after the MNPs@OLAC-MH nanocomposite obtained after the MH drug was bound to the surface of the MNPs@OLAC nanocomposite is due to the irregular distribution of the drug in the surface morphology and pore structures of the nanocomposite (Baig et al., 2022).

The DLS analysis results of MNPs@OLAC and MNPs@OLAC-MH nanocomposites are shown in Figure 3. Analyses were performed with the solution obtained by weighing 1 mg of the nanocomposite material and sonicating it in 10 mL of deionized water. When the results of the DLS analysis are examined, it is seen that MNPs@OLAC and MNPs@OLAC-MH nanocomposites have an average size distribution of 122 and 190 nm, respectively (Kovrigina et al., 2022).

The zeta potential analysis results of MNPs@OLAC and MNPs@OLAC-MH nanocomposites are shown in Figure 4. As a result of zeta potential measurement, information about the electric charge on the surface of any material is obtained. To prevent agglomeration of MNPs@OLAC and MNPs@OLAC-MH nanocomposites and to ensure stability between nanocomposites, it is desired to obtain either high positive or negative zeta potential values. The potential values of MNPs@OLAC and MNPs@OLAC-MH nanocomposites under the determined optimum conditions were measured as −16.8 and −16.7 mV, respectively. When the results are examined, it is seen that MNPs@OLAC and MNPs@OLAC-MH nanocomposites show low agglomeration due to their negatively charged value, and therefore the prepared suspensions maintain their stability for a long time (Attia et al., 2022).

At a temperature of 37°C, Figure 5 illustrates the cumulative release of MH in MNPs@OLAC at pH 7.4 (the physiological pH) and pH 5.4 (the acidic environment in tumor tissues). MH was found to be released more under physiological settings, and the release of MH changed determined by the pH value that was evaluated. This phenomenon was discovered when different pH values for MH release were investigated (Cunha et al., 2023). To perform the release studies, 10 mL solutions of 96 μg/mL MH loaded into MNPs@OLAC at different pH values and concentrations were used. The ratio of MR mass to the volume of the release medium was found to be 9.6 μg/mL. After 24 h, the release from bulk MH decreased from 48.98% at pH 7.4%–13.47% at pH 5.4. At pH 5.4, in the release of loaded MH in MNPs@OLAC, the release was 10.86% after 6 h, 12.48% after 12 h, 14.89% after 48 h, and 15.02% after 72 h (Kulkarni and Belgamwar, 2019; Jangid et al., 2020). At pH 7.4, in the release of loaded MH in MNPs@OLAC, the release was 45.04% after 6 h, 47.00% after 12 h, 50.44% after 48 h, and 53.01% after 72 h.

MCF-7 breast cancer model (Fuster et al., 2021), U373, HT-29 colon model (Singh et al., 2022), glioblastoma model (Jiang et al., 2024) and T98-G glioblastoma model (Long et al., 2022) cell lines have been widely used as in various studies. Furthermore, HUVEC was utilized as a healthy cell line throughout the course of this research. The MTT assay was utilized in order to examine the cytotoxic effects of MH loaded in MNPs@OLAC nanocomposites against the cell lines HT-29, MCF-7, U373, T98-G, and HUVEC.

Through the process of reduction, a tetrazolium compound (MTT) is transformed into formazan in the MTT test, which is a colorimetric assay that is extensively used (Lovato et al., 2024). For the purpose of determining cellular metabolic activity, which serves as an indicator of toxicity, colorimetric tests are utilized extensively (Abd AL-Hadi et al., 2024). The effect of MH release in pH 7.4 and 5.4 solutions of MNPs@OLAC-MH prepared in phosphate buffer on cell viability of HT-29, MCF-7, U373, T98-G, and HUVEC cell lines was evaluated after 24 and 48 h. Interaction with MH-loaded MNPs@OLAC nanocomposites at pH 7.4 for 24 h (Figure 6) did not cause significant differences in the viability of tested HT-29, MCF-7, U373, and T98-G cells and was significantly above the concentration ranges of tested MNPs@OLAC-MH for 24 h. However, as seen in Figure 6, after 48 h of interaction, 50 ug/mL concentration and T98-G showed an effect on cell viability.

Effect of MNPs@OLAC-MR (1–200 μg/mL prepared with PBS 7.4 buffer) on cell viability of HUVEC cell line, T98-G cell line, MCF-7 cell line, U373 cell line, and HT-29 cell line. Cell viability was assessed using MTT assay after 24 and 48 h of exposure.

When exposed to MH-loaded MNPs@OLAC nanocomposites at pH 5.4 for 24 and 48 h, the viability of the evaluated HT-29, MCF-7, U373, and T98-G cells did not show any significant alterations (Figure 7). The concentration ranges of MNPs@OLAC-MH at 24 and 48 h were notably greater than the concentration ranges determined for the tested MNPs@OLAC-MH.