# Reimagine breast cancer treatment: MgO-IDA nanocomposites: A promising strategy for precision medicine targeting MCF-7 cells

Shoaib Shafique, Asma Irshad, Uzair Ishtiaq, Lotfi Mhamdi, Sumaira Sharif, Rabbia Jawad, Patrick Martin, Naceur M’Hamdi, Saad Arshad, Bochra Bejaoui

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Shoaib Shafique, Asma Irshad, Uzair Ishtiaq, Lotfi Mhamdi, Sumaira Sharif, et al.. Reimagine breast cancer treatment: MgO-IDA nanocomposites: A promising strategy for precision medicine targeting MCF-7 cells. Results in Chemistry, 2025, 17, 10.1016/j.rechem.2025.102660. hal-05421865

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# Reimagine breast cancer treatment: MgO-IDA nanocomposites: A promising strategy for precision medicine targeting MCF-7 cells

<figure><img src="image_2.png" /></figure>

Shoaib Shafique<sup>a</sup>, Asma Irshad<sup>a, *,</sup>, Uzair Ishtiaq<sup>b, c, **</sup>, Lotfi Mhamdi<sup>d, *</sup>, Sumaira Sharif<sup>e</sup>, Rabbia Jawad<sup>f</sup>, Patrick Martin<sup>g</sup>, Naceur M’Hamdi<sup>h</sup>, Saad Arshad<sup>i</sup>, Bochra Bejaoui<sup>j, k</sup>

## ARTICLE INFO

### Keywords:

Anticancerous drug

MgO-IDA nanocomposite

Targeted drug delivery

Controlled drug release

In vivo studies

## ABSTRACT

Conventional chemotherapies are used to treat cancer; however, they cannot discriminate between healthy and tumor tissues. Currently, advanced drug delivery systems are being developed to overcome these challenges. The present study aims to design a targeted and controlled drug delivery system against cancer tissues. To achieve this goal, MgO nanoparticles were synthesized via the sol-gel method, conjugated with IDA and folic acid, and then characterized via UV-Vis, FT-IR, SEM-EDX, and XRD techniques. The anticancer potential of the nanocomposite was subsequently evaluated through an MTT assay on the MCF-7 cell line, a migratory assay for cell metastasis, histochemical analysis, a drug release assay, and a hemolysis assay to assess biosafety. Furthermore, in vivo toxicity studies were performed on *Mus musculus* (albino mice) to evaluate the toxicity of different concentrations of MgO-IDA. The results were statistically analyzed via one-way ANOVA and are presented as percentages. The white MgO and purple MgO-IDA nanocomposites presented UV-Vis absorption peaks at 295 and 445 nm, respectively. The FT-IR spectra confirmed the successful conjugation of MgO and MgO-IDA through the appearance of peaks at 490 cm⁻¹ and 700 cm⁻¹. SEM analysis revealed a spherical morphology and uniform structure of the nanoparticles, and EDX analysis confirmed the composition, with significant peaks between 0.2 and 1.5 keV and at 2.55 keV. XRD analysis of MgO-IDA revealed diffraction peaks corresponding to the (111), (200), (220), (311), (422), (400), and (440) planes. Compared with IDA, MgO-IDA exhibited significant cytotoxic effects on the MCF-7 cell line and a reduced cell metastasis rate. Histochemical analysis revealed significant binding of MgO-IDA to folate receptors in breast cancer tissue. Additionally, enhanced release of IDA from MgO-IDA was observed in the presence of proteinase K. A hemolysis assay revealed less than 3.5 % hemolytic activity, confirming the biocompatibility of the nanocomposite with human RBCs. In vivo, studies have also revealed the nontoxic behavior of MgO NPs at a concentration of 250 mg kg⁻¹ and that of MgO-IDA at 100 mg kg⁻¹. These results indicate that the synthesized nanocomposite is safe and efficient as a targeted drug delivery system for breast cancer therapy.

<footnote><sup>a</sup> School of Biochemistry and Biotechnology, University of the Punjab, Pakistan</footnote>

<footnote><sup>b</sup> Department of Research and Development, Paktex Industries, Kamoke, 52470, Pakistan</footnote>

<footnote><sup>c</sup> Department of Life Sciences, University of Management and Technology, Lahore, Pakistan</footnote>

<footnote><sup>d</sup> University de Franche-Comté. STAPS Besançon, UMR Right, TAI-IT, EPSI, College of Medicine Besançon, 25030, France</footnote>

<footnote><sup>e</sup> Institute of Molecular Biology and Biotechnology, The University of Lahore, Pakistan</footnote>

<footnote><sup>f</sup> School of Biological Sciences, University of the Punjab, Pakistan</footnote>

<footnote><sup>g</sup> Univ. Artois, Unilasalle, ULR7519 - Unité Transformations &Agro-Ressources, F-62408, Béthune, France</footnote>

<footnote><sup>h</sup> University of Carthage, National Agronomic Institute of Tunisia, Research Laboratory of Ecosystems & Aquatic Resources, 43 Avenue Charles Nicolle, Tunis 1082, Tunisia</footnote>

<footnote><sup>i</sup> Department of General Surgery, Siddique Family Hospital, Pakistan</footnote>

<footnote><sup>j</sup> University of Carthage, National Institute of Research and Physico-Chemical Analysis, Technopark of Sidi Thabet, Laboratory of Useful Materials, Ariana 2020, Tunisia</footnote>

<footnote><sup>k</sup> University of Carthage, Faculty of Sciences of Bizerte, Department of Chemistry, Bizerte 7021, Tunisia</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author at: Department of Life Sciences, University of Management and Technology, Lahore, Pakistan.</footnote>

<footnote>E-mail addresses: uzair.saroya9@gmail.com (U. Ishtiaq), lotfi.mhamdi@univ-fcomte.fr (L. Mhamdi).</footnote>
# 1. Introduction

Nanotechnology has unlocked transformative methods in many areas of scientific research, especially medical sciences [1–3], and revolutionized approaches to therapeutics, diagnostics, and various regenerative medications [4,5]. At the core of nanotechnology, the targeted drug delivery system is the most significant behavior of nanoparticles [6]. Traditional medications and techniques often struggle to diagnose cancer in its early stages, as cancer typically presents symptoms only in its advanced stages, which reduces the chance of successful treatment and increases the risk of mortality [7,8]. A variety of anticancer drugs, including chemotherapy, are employed in different stages of cancer treatment. Idarubicin is a commonly used chemotherapeutic agent in the treatment of breast cancer [9]. However, this drug often induces cell death through the inhibition of cell proliferation and the production of reactive oxygen species (ROS). This drug helps inhibit cancerous cell growth by intercalating cellular DNA and damaging the cellular content (Fig. 1c) [10,11]. However, it has been reported that this drug inhibits the proper unwinding of DNA, a crucial step during replication and transcription [12]. Although idarubicin and related chemotherapeutic drugs can degrade tumor cells, their anticancer potential and extensive off-target effects on healthy tissues are often limited [13]. These drugs also show less solubility and instability in biological systems, which results in more complex conditions for treating tumors [12]. They are nonspecifically distributed throughout the body and cause damage to healthy tissues [14]. Although various literature studies have reported the anticancer and drug targeting effect of MgO nanoparticles while its efficacy in conjugation with extensively used chemotherapeutic drug i.e. Idarubicin has never been investigated. Moreover, multiple side-effects of Idarubicin in biomedical applications are also mentioned in existing publications i.e. severe leukopenia with infection and bleeding, thrombocytopenia, asymptomatic LVEF fall to over heart failure, tissue necrosis, diarrehea, vomiting, nausea, alopecia, etc. [15].

Nanotechnology offers promising nanocarriers, such as dendrites, liposomes, polymeric nanoparticles, and solid lipid nanoparticles, which can deliver drugs that are precisely targeted to tumor sites [16]. These nanocarriers are designed with specific ligands that bind to receptors on targeted tumor sites, allowing highly targeted drug delivery [17]. For an

effective nanobased drug delivery system, two main approaches are employed: active and passive delivery, which are adopted by different nanoparticles, depending on their properties. An active drug delivery system (Fig. 1a) involves interactions between ligands on drug nanocomposites and receptors present on tumor cells [18]. Nanobased drugs such as conjugated MgO nanoparticles are designed with drugs, nucleic acids, or special signaling molecules attached to their surface that specifically target receptors, i.e., tumor markers, peptides, or antibodies, on the surface of melanoma cells [19,20]. In site-specific and controlled drug delivery, the tumor's external environment is also a crucial factor in regulating the drug delivery system. Tumor tissues are subjected to high pressure, increased gene expression, low pH, and low oxygen levels. These factors are also important for diligently inducing drugs specifically to the target site. Owing to the slightly acidic pH inside tumor cells, the drug nanocomposite disintegrates, and the drug, i.e., IDA, enters the nucleus, where it interacts with mutated DNA (Fig. 1b) [21,22].

In addition to ligand-receptor binding, a passive delivery system also helps to target the tumor sites present in the human body [19]. As tumors grow, they oppose the body's normal circulatory system, and to meet their nutritional requirements, these cells establish their circulatory system for suitable nutrients and growth factors [23,24]. This self-angiogenesis creates uneven angiogenetic signals and irregular vascular networks in malignant tissues, ultimately resulting in the loss of structural integrity and hence subsequent damage to the endothelial barrier [25]. Such tumor sites have irregular gaps with no proper lymphatic flow, which allows different components to stay at these sites longer [26,27]. Drug nanocomposites are specifically designed so that they can easily pass through normal tissues and be collected in the gaps of melanoma tissues for a longer period [28].

Various metals and metal oxide nanoparticles are widely used for specific drug delivery to tumor cells. Their stable and robust structure protects sensitive drugs from enzyme degradation [29]. Magnesium oxide (MgO) and zinc oxide (ZnO) nanoparticles provide suitable protective shells for sensitive drugs, ensuring their stable transport to the target site [30,31]. The high surface area-to-volume ratio allows enhanced absorption of hydrophobic drugs, which are usually difficult to access via traditional methods. Magnesium oxide (MgO)

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Active drug delivery system and anticancer potential mechanism of MgO nanoparticle-conjugated idarubicin: (a) Receptor-mediated endocytosis of drug nanocomposites via specific receptors on the surface of cancer cells; (b) disintegration of drug nanocomposites due to the slightly acidic environment inside cancer cells; (c) interaction of idarubicin with mutated DNA in the nucleus and inhibition of proliferation.</figcaption></figure>
nanoparticles can respond to specific stimuli, i.e., a magnetic field, pH, and temperature, which make them more beneficial for specific drug targeting [32]. Cancer cells reside in an acidic tumor microenvironment, which can trigger pH-sensitive magnesium oxide and zinc oxide nano-particles, leading to more targeted and less toxic cancer therapies [33,34].

The novelty of current research work is the synthesis and characterization of folic acid (FA) attached magnesium oxide nanocomposite to especially conjugate idarubicin in order to reduce its off-target effects of anticancer drug and malignancy of cancerous cells in breast cancer treatment. The comparison of bare IDA and MgO-IDA nanocomposite is also mentioned in respect to decrease the metastasis behavior of breast cancer cells in the presence of both Idarubicin and MgO-IDA nanocomposite. By leveraging the therapeutic properties, such as biocompatibility, low toxicity, and stability, of MgO nanoparticles, this research sought to reduce the off-target effects associated with the increased anticancer potential of idarubicin. Through the combination of in vitro and in vivo studies, this study evaluated the anticancer and biosafety potential of MgO nanoparticles in MgO-IDA nanocomposites, highlighting the use of MgO nanoparticles as potent and safe delivery vehicles to treat breast cancer.

## 2. Materials and methods

The chemicals and reagents used in this study were obtained from Bio-Rad Laboratories and Sigma Aldrich (Merck KGaA) (USA), and an anticancer drug such as idarubicin was purchased from Al-Habib Pharmaceuticals, Pakistan. The research work was conducted at the Molecular Nanotechnology Lab, School of Biochemistry and Biotechnology, University of Punjab, Lahore, Pakistan.

### 2.1. Synthesis and characterization of MgO nanoparticles

The magnesium oxide MgO nanoparticles were synthesized via a modified sol-gel technique, following the procedure described by Salman, K.D. et al. [35]. A 0.2 M solution of magnesium nitrate hexahydrate ($MgNO_3 \cdot 6H_2O$) (CAS No: 13446-18-9, 99 % ACS reagent, Sigma-Aldrich, USA) and a 0.5 M solution of sodium hydroxide (NaOH, CAS No. 1310-73-2, ≥98 % ACS reagent grade) were prepared in 100 mL of deionized water. The NaOH solution was added dropwise to the $MgNO_3$ solution with continuous stirring for 5 min until white precipitates formed. After stirring for an additional 30 min, the pH was adjusted to 12.5, and the solution was then centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and the pellet was washed 3-6 times with methanol and deionized water and then dried at room temperature (Fig. 2). The obtained dried powder was then calcined at 500 °C for 3 h. The resulting MgO nanoparticles were characterized via a Shimadzu UV-1900i UV-Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan) in the wavelength range of 190-430 nm.

### 2.2. Synthesis of BSA-coated MgO nanoparticles

To enhance stability and biocompatibility, synthesized MgO nanoparticles have been coated with bovine serum albumin (BSA), which prevents them from aggregating [36]. A mixture of 80 mg of BSA (CAS No. 9048-46-8, 99 % fraction V, protease-free, Sigma-Aldrich, USA) and 20 mg of MgO nanoparticles was added to 25 mL of deionized water and stirred at room temperature for 15 min. The successful coating of MgO nanoparticles with BSA was confirmed by a ninhydrin test. BSA-coated MgO nanoparticles were added to the sterile test tube, followed by the addition of 0.5 mL of 1 % ninhydrin (CAS No. 485-47-2, ≥98 % ACS reagent grade, Sigma-Aldrich, USA) solution. The test tube was heated in a boiling water bath for 5 min, and a color change was observed (Fig. 2) [37].

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Schematic diagram showing the synthesis of MgO nanoparticles followed by multiple steps, including BSA coating to MgO nanoparticles, IDA conjugation, and folic acid attachment, ultimately leading to the MgO-IDA nanocomposite.</figcaption></figure>
## 2.3. Synthesis of idarubicin-conjugated BSA–MgO nanoparticles and folic acid attachment

Idarubicin (IDA), an anticancer drug, was conjugated with BSA-MgO nanoparticles via the carbodiimide method, with slight modifications to the procedure described in reference [38]. First, 10 mg of idarubicin dissolved in DMSO (dimethyl sulfoxide, CAS No. 67–68.5, ≥99.9 % purity (ACS grade), Merck (Germany)) was added to the BSA-MgO nanoparticles, and the pH was then adjusted to 8.0 with 1 N NaOH. After that, 400 mg of EDAC (EDC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, CAS No. 25952–53-8) from Bio-Rad Laboratories (Catalog No. 1530990) was added to the solution, and the pH was adjusted again to 6.0 with 1 N HCl. The solution was stirred at room temperature for 6 h, after which an additional 400 mg of EDC was added. The MgO-IDA nanocomposite was then kept in the dark with continuous stirring at room temperature for 15 h.

Furthermore, folic acid was conjugated to the nanocomposite via the glutaraldehyde method [39]. Glutaraldehyde was added to the drug-conjugated BSA–MgO composite to a final concentration of 5 %. The mixture was incubated at room temperature for 3 h with stirring at 100 rpm. 5 mg folic acid (CAS No. 59–30-3, 98 % purity, Sigma-Aldrich (USA)) was added, and then the mixture was incubated for 24 h at room temperature with stirring at 100 rpm. The nanocomposite was centrifuged for 15 min at 7000 rpm, washed with a buffer solution (0.01 M Tris and 0.15 M NaCl (CAS No. 7647-14-5, ≥99.0 % ACS reagent grade, Sigma-Aldrich, USA)) and finally dried for 48 h at 40 °C (Fig. 2).

## 2.4. Characterization of the idarubicin-conjugated MgO nanocomposite

**UV–Vis spectroscopy:** The concentrations of the MgO nanoparticles and MgO-IDA nanocomposite were determined via UV–Vis spectroscopy. Both nanoparticles were separately dissolved in deionized water, and their UV–Vis spectra were obtained in the wavelength range of 190–430 nm [40] via a Shimadzu UV–1900i UV–Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

**Fourier transform infrared (FT-IR) spectroscopy:** The functional groups and chemical bonds present in the MgO-IDA nanocomposite before and after conjugation were determined via FT-IR. Spectra were obtained in dry air at room temperature [41] via a ThermoFisher-Scientific Nicolet iS50 spectrometer (Thermo Fisher Scientific, USA) with a potassium bromide (KBr) pellet in the wavelength range of 400–4500 cm⁻¹.

**Scanning electron microscopy-energy dispersive X-ray (SEM-EDX):** The surface morphology and elemental analysis of the MgO-IDA nanocomposite were obtained via SEM and EDX, respectively. Scanning electron microscopy was performed at different magnifications (100×, 250×, 500×, and 1500×) via a Nova NanoSEM 450 analyzer (Thermo Fisher Scientific, USA) to visualize the nanoparticle morphology, and energy dispersive X-ray spectroscopy (EDX) was performed to obtain the composition of the nanocomposite [40].

**X-ray diffraction (XRD):** The crystalline structure and phase purity of the MgO-IDA nanocomposite were determined via XRD. A Bruker AXS D8 Advance diffractometer (Bruker Corporation, Karlsruhe, Germany) equipped with a copper anticathode (Cu K = 1.5402 Å) in the 2θ range between 10° and 90° with a step size of 0.0101/min was used [40]. The crystalline phases present in the sample were identified via the American Society for Testing and Materials (ASTM) database [40]. The particle size was calculated from the FWHM of the diffraction peaks via the Scherrer eq. (1):

$$D = \frac{K \lambda}{\beta \cos\theta} \qquad (1)$$

where K is a constant, λ is the X-ray wavelength, β is the full width at half maximum (FWHM) of the diffraction peak, and cosθ is the diffraction angle.

## 2.5. Anticancer potential of the MgO-IDA nanocomposite

### 2.5.1. MTT assay

For the MTT assay, MCF-7 breast cancer cell lines were cultured at 37 °C in MEM medium (10 % FBS, 1 % Penicillin-Streptomycin solution) in humidified atmosphere of 5 % CO₂ followed by digestion 0.25 % trypsin containing EDTA to get single cell suspension. 5000 cells/well were seeded in a Thermo Scientific™ Nunc™ 96-well MicroWell™ and allowed to reach 70–80 % confluency. Various concentrations (4–24 µg/ mL) of MgO, IDA, and the MgO-IDA nanocomposite were then added to the wells in triplicate followed by incubation at 37 °C in a cell incubator of 5 % CO₂ and > 90 % humidity for 24 h, while some of the wells were left untreated to serve as a baseline for viability. A 5 mg/mL stock solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was prepared in phosphate-buffered saline (PBS) and then diluted to 10 %. After 24 h of incubation, the treatment media was discarded; yellow-colored MTT reagent was added to the wells, which were subsequently incubated for 3 h in a humidified atmosphere with 5 % CO₂. By observing purple crystal formation, the MTT solution was carefully removed without disturbing the formed purple crystals. 100 µL DMSO was then added to dissolve the crystals, and the plates were placed in the dark for 20 min at room temperature. The protocol was followed by ISO standard method: ISO 10993-5: 2009 (Protocol number: SST22040422819BB) obtained from CCIC Huatongwei International Inspection(Suzhou)Co., Ltd. The absorbance of each well was subsequently measured at 570 and 650 nm via a plate reader.

### 2.6. Cell migration assay

The effects of the MgO, IDA, and MgO-IDA nanocomposites on the cellular migration activity were further assessed. The protocol was adapted from [42] with minor modifications. A total of 0.4 million MCF-7 cells (Sigma–Aldrich, Merck, Germany) were seeded in four separate 6-well plates and incubated for 24 h to reach 100 % confluency. A p200 micropipette was then used to scratch the center of the well, and the media was removed. The wells were washed with 1× PBS to remove detached cells from the scratch area. The MgO, IDA, and MgO-IDA nanocomposites were then separately added to triplicate wells, while the untreated wells served as controls for the baseline measurements. Then, the control wells were treated with Dulbecco’s modified Eagle’s medium (DMEM) for 48 h. Images were taken from the scratch area at different time intervals (0, 6, 24, and 48 h) for morphological analysis.

### 2.7. Histochemistry (HC) of BC

The localization, binding, and therapeutic efficacy of the MgO-IDA nanocomposite were evaluated through histochemical analysis of BC samples. The tissue samples were cut into 4 mm small sections and placed in 10 % sucrose solution (sucrose, TBS buffer) for dehydration. The samples were incubated for 10 h in a shaker, after which the sucrose solution was removed. This step was repeated with various concentrations (15, 25, and 35 %) of sucrose solution to obtain complete dehydration of the tissues. These tissues were wrapped in aluminum foil molds at the optimum cutting temperature (OCT) and kept at –80 °C for 48 h. For fixation, a sharply cut section of frozen tissue was placed on an albumin-coated slide and stored at –80 °C for experimentation. One drop of 5 % BSA was added to the slide to block the tissue section, which was subsequently incubated for 1 h. Then, the slides were washed and treated with the MgO-IDA nanocomposite with further incubation for 1 h. All the treated slides were observed under an Olympus BX43 fluorescence microscope (Olympus Corporation, Tokyo, Japan) at 40× magnification. This experiment was performed according to the instructions of Mubeen et al. [43].
## 2.8. Drug release study

The release behavior of idarubicin from the MgO-IDA nanocomposite was assessed through a dialysis-based drug-releasing method (DM). The protocol followed the guidelines of Elahi Attari et al. [44] with slight modifications. This work evaluated the drug-releasing activity of the enzyme proteinase K at pH 5.4. For this purpose, 5 mg of the MgO-IDA nanocomposite and 5 mg of proteinase K were dispersed in 2 ml of PBS, the solution was collected in a dialysis bag, and the mixture was immersed in 250 mL of PBS at pH 5.4. The mixture was placed on a shaking incubator at 37 °C for 4 days. Then, with a specific incubation, 2 ml of fresh PBS was added instead of dialysate to stimulate lysosomal conditions (with proteinase K at pH 5.4). The efficiency of idarubicin release was analyzed at different time intervals via a UV-Vis spectrophotometer at wavelengths ranging from 450 to 480 nm. All the experiments were performed in triplicate to ensure the reproducibility of the results.

## 2.9. Biosafety analysis of the MgO-IDA nanocomposite

### 2.9.1. Hemolysis assay

Blood samples were centrifuged at 3000 rpm for 15 min to separate human red blood cells from other blood components. The obtained pellet containing RBCs was washed with PBS, and 1 mL of the MgO-IDA nanocomposite (10 mg/mL) was added to 1 mL of RBCs and incubated at 37 °C for 4 h in a shaking incubator. One milliliter of deionized water and 1 mL of PBS were used as positive and negative controls, respectively. After incubation, the samples were centrifuged at 13000 rpm for 15 min, and the supernatant was collected and analyzed via a UV-Vis spectrophotometer at a wavelength of 540 nm [1,44]. The experiment was conducted in triplicate, and the percentage hemolysis of RBCs was calculated via formula (2).

$$ \%hemolysis = \frac{Treated sample - Negative control}{Positive control - Negative control} \times 100 \quad (2) $$

## 2.10. In vivo studies: toxicity analysis of the MgO-IDA nanocomposite

To investigate the safety and biocompatibility of the MgO-IDA nanocomposite with the body's vital organs, toxicity analysis was conducted using albino mice. All animal experiments were conducted with the explicit approval of the Paktex Industries Institutional Animal Care and Use Committee (IACUC), under protocol number PI/QC/202/24/PUW. All experimental procedures and animal care adhered strictly to the guidelines and regulations set forth by Paktex Industries IACUC, which are in full compliance with recognized ethical principles for animal research. Furthermore, all methods and reporting were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org) to ensure transparency and reproducibility. The selected animals were managed under following conditions i.e. feed comprising full-price pellets, Pizhou Xiaohu Technology Development Co., Ltd., drinking water met the Standards for Drinking Water Quality GB 5749-2006, at room temperature 20-25 °C, humidity 30%-70%, under 12 h light/dark cycle, full-spectrum lighting. The mice were divided into 11 groups: comprising 10 experimental groups and one control group. The control group were included the healthy mice with no administration of drug. Groups 1-5 received intravenous MgO NPs doses of 50, 100, 250, 350, and 500 mg kg⁻¹, respectively while groups 6-10 were administrated MgO-IDA nanocomposite intravenously with 25, 50, 100, 150, and 200 mg kg⁻¹, respectively. All the doses were prepared in water. Toxicity analysis involved close monitoring of various physiological and behavioral parameters i.e. pupil size, exophthalmos, piloerection, writhing, respiratory patterns, convulsions, aggression, hypersensitivity, salivary secretions, lacrimation, skin discoloration, prehensile strength, hypothermia, and abnormal behavior.

## 2.11. Statistical analysis

IBM SPSS Statistics 28 was used to perform the statistical analysis. The obtained values were interpreted via one-way ANOVA, and the results are presented as percentages (%). Graphical representations, including bar charts, have been mentioned to better summarize the findings of this study.

## 3. Results and discussion

NPs are used as a specific drug delivery system in the treatment of various fetal diseases [4]. Anticancer drugs, i.e., chemotherapy, treat tumor cells via different mechanisms, but they often have off-target effects on healthy tissues in the body [13]. This approach reduces the efficacy of anticancer drugs because of their low solubility, instability, and nontargeting nature. Additionally, such medications are not fully transported to the tumor site and are unable to completely degrade melanoma cells [12]. Metal oxide nanoparticles, such as magnesium oxide (MgO), offer a potential solution because of their stability, non-toxicity, and large surface area-to-volume ratio. All these properties allow them to be safe and stable hydrophobic drug delivery systems for the treatment of breast cancer.

### 3.1. Synthesis of MgO, BSA-MgO, and MgO-IDA nanocomposites

The MgO nanoparticles were synthesized via the sol-gel technique, which involves a chemical reaction between magnesium nitrate hexahydrate and sodium hydroxide. The white powdered MgO nanoparticles (Fig. 3a) were coated with BSA to functionalize them for binding with the idarubicin drug and folic acid. The successful binding of BSA to MgO nanoparticles was confirmed through the ninhydrin test. BSA, being a protein, will bind with ninhydrin if present in the solution, resulting in an indication of the attachment of BSA to the surface of MgO nanoparticles.

The binding of BSA with MgO nanoparticles provided functional groups, such as carboxylic acid and amino groups, for the further attachment of idarubicin and folic acid molecules. Idarubicin was bound to the nanoparticles via the carbodiimide method, whereas folic acid was attached via the glutaraldehyde method. Folic acid acts as a signaling molecule that targets the folate receptors of cancer cells. Following the complete experimental protocol, a fine powder of the MgO-IDA nanocomposite was obtained (Fig. 3b) and stored for in vitro and in vivo studies.

### 3.2. Characterization of MgO and MgO-IDA nanocomposites

#### 3.2.1. UV-Vis spectroscopic analysis of MgO and MgO-IDA nanocomposites

The concentrations of the MgO nanoparticles and idarubicin-conjugated MgO nanocomposite were determined via UV-Vis

<figure><img src="image_5.png" /><figcaption>Fig. 3. Powder of (a) MgO nanoparticles and (b) idarubicin-conjugated MgO nanoparticles.</figcaption></figure>
spectroscopy at wavelengths ranging from 190 to 430 nm. The UV–Vis spectrum displayed a significant peak at 295 nm, confirming the synthesis of MgO nanoparticles, as shown in Fig. 4a. MgO nanoparticles have been reported to exhibit various peaks in the range of 250–310 nm, depending on their synthesis source. Moreover, the optical properties of MgO nanoparticles are influenced by their size and shape [45,46]. The literature has reported the synthesis of MgO nanoparticles from *Pterocarpus marsupium* rox. b Heartwood extract, and the results showed a significant peak at 310 nm, indicating the presence of MgO nanoparticles in the sample [47]. In a similar study conducted by Muhammad Isa Khan et al. [48], MgO nanoparticles were prepared from *Dlabergia sissoo* leaf extract. Their results revealed a significant peak at 268 nm, confirming the successful synthesis of MgO nanoparticles [48]. In the present study, MgO nanoparticles were characterized via UV–Vis spectroscopy and then conjugated with the drug idarubicin. This conjugate was further analyzed through UV–Vis spectroscopy, and the obtained spectrum showed peaks at 310 and 445 nm, confirming the presence and attachment of idarubicin on the surface of the MgO nanoparticles (Fig. 4b).

### 3.3. Fourier transform infrared (FT-IR) spectroscopy analysis of the MgO and MgO-IDA nanocomposites

The functional groups and chemical bonds present before and after the conjugation of the idarubicin drug with MgO nanoparticles were assessed via FT-IR spectroscopy. Fig. 5a shows the functional groups present in the structure of idarubicin [49], and Fig. 5b displays the FT-IR spectrum, showing various peaks at 4901324 and 3690 cm⁻¹. The strong, sharp peak obtained at 1324 cm⁻¹ is attributed to the bending vibrations of water molecules on the surface of the MgO nanoparticles, whereas the weak peaks at 490 cm⁻¹ and 3690 cm⁻¹ indicate the presence of Mg–O vibrations and the O–H stretching mode of the hydroxyl group, respectively. The peaks at 490 and 3690 cm⁻¹ were attributed to the presence of moisture on the surface of the nanoparticles. Various methods can be used to synthesize MgO nanoparticles, such as the microwave method and sol–gel method. Khansa D. Salman et al. used these two methods for the preparation of MgO nanoparticles and characterized them via various techniques, including XRD, FT-IR, and FESEM. The FT-IR results revealed similar peaks at three different wavelengths: 424, 1423, and 3695 cm⁻¹ when the microwave method was used and 413, 1454 and 3672 cm⁻¹ when the sol–gel method was used [35]. The synthesis method can significantly influence the functional groups and bonding present in nanoparticles. After conjugation with an anticancer drug, FT-IR analysis of the nanocomposite revealed six peaks at 490, 700, 1400, 1633, 2443, and 3690 cm⁻¹, as shown in Fig. 5c. Two peaks at 490 and 3690 cm⁻¹ were observed in the FT-IR spectrum of the MgO nanoparticles, corresponding to MgO bonds and hydroxyl groups, respectively. Additional peaks were observed in the

spectrum, which can be attributed to the presence of idarubicin, BSA, and folic acid. The peak at 700 cm⁻¹ indicated the presence of C–H bending vibrations associated with the folic acid and aromatic rings of idarubicin. The peak at 1400 cm⁻¹ is assigned to C–O stretching vibrations from the carboxylic acid groups of folic acid and BSA, whereas the peaks at 1633 cm⁻¹ and 2443 cm⁻¹ strongly depict the presence of idarubicin in the sample, as the peak at 1633 cm⁻¹ is due to C=O stretching vibrations of carbonyl groups prominent in the idarubicin structure due to the presence of ketone rings in its anthracyclines. The peak at 2443 cm⁻¹ was attributed to the C–H stretching of methyl groups present in the aromatic rings and alkyl chains of the idarubicin structure. These spectral features strongly support the successful conjugation of anticancer drugs to MgO nanoparticles.

### 3.4. Scanning electron microscopy-energy dispersive X-ray (SEM-EDX) analysis of the MgO-IDA nanocomposites

To investigate the surface morphology, structure, agglomeration, and elemental composition of the MgO-IDA nanocomposite, scanning electron microscopy and energy dispersive X-ray studies were performed. As shown in Fig. 6a, smooth spherical shaped MgO nanoparticles have shown with particle size 20–25 nm in the range of scale bar 100 µm while after conjugation with Idarubicin and Folic acid attachment the SEM analysis showed irregular, blocky and heterogeneous morphology of MgO-IDA nanocomposite (Fig. 6b). The size and shape of nanocarriers greatly influence the delivery of drugs to target sites. The MgO nanoparticle size obtained in this study is greater than that reported in the literature because of the attachment of the drug on the surface of the nanoparticles. Furthermore, the EDX profile showed significant peaks at approximately 0.2–1.5 and 2.55 Kev, revealing the successful formation of MgO-IDA nanocomposites (Fig. 7). Various other elements, including carbon, magnesium, oxygen, and hydrogen, were also detected (Table 1). The peak obtained at 1.5 keV indicates the presence of MgO nanoparticles, whereas the peaks below this value indicate the presence of idarubicin, BSA, and folic acid molecules.

Fouda et al., 2021 [50] biologically synthesized MgO nanoparticles and determined their structural morphology through elemental analysis, which aligned with the results of the present study. Similarly, other studies have assessed the morphological characteristics of MgO nanoparticles and reported a uniform spherical structure [51,52]. These studies revealed the structure and morphology of MgO nanoparticles, which may differ slightly in the current work because of the conjugation of MgO nanoparticles with anticancer drugs. In this study, the elemental analysis revealed different peaks at approximately 0.2–0.4 Kev in the EDX profile, which are not present in the literature.

<figure><img src="image_6.png" /><figcaption>Fig. 4. UV–Vis spectra of the (a) MgO nanoparticles and (b) MgO-IDA nanocomposite.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Fig. 5. (a) The structural formula of idarubicin shows various functional groups, (b) the FT-IR spectrum contains peaks for MgO nanoparticles, and (c) the FT-IR spectrum contains peaks for MgO-IDA nanocomposites.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 6. Morphology of the (a) MgO NPs and (b) MgO-IDA nanocomposite obtained by scanning electron microscopy (SEM).</figcaption></figure>

### 3.5. X-ray diffraction (XRD) analysis of the MgO-IDA nanocomposites

The phase purity of the MgO-IDA nanocomposites was determined through X-ray diffraction analysis. Fig. 8 shows various peaks indicating the presence of both MgO and IDA in the sample. In Fig. 8a, various broad and sharp peaks were obtained for MgO nanoparticles which indicated small size cubic MgO nanoparticles, in accordance with the JCPDS card (JCPDS card, No. 45–0946) while Fig. 8b showed seven characteristic peaks of MgO, BSA, folic acid, and idarubicin at 2θ values of 28°, 33°, 39°, 48.5°, 52°, 57°, and 64°, corresponding to indices of 111, 200, 220, 311, 422, 400, and 440, respectively (Table 2). The peaks at 2θ values of 111, 200, 220, and 311 are related to the presence of MgO nanoparticles, whereas the peaks at 422, 400, and 440 are associated with the presence of idarubicin attached to the surface of the nanoparticles. Compared with the literature, MgO nanoparticles present four distinct peaks with plane indices of 111, 200, 220, and 311, as reported by various scientists [51,53]. Almontasser et al. [54]. MgO nanoparticles were synthesized via the precipitation method, and the phase purity of the nanoparticles was analyzed via X-ray diffraction (XRD). These genes presented four peaks that coincided with those observed in the present

study. Additionally, the cellular activity of idarubicin-conjugated magnetic nanoparticles was studied in the HL-60 cell line [55]. The authors synthesized glutaraldehyde-coated magnetic nanoparticles conjugated with idarubicin and characterized them via various methods. The XRD pattern revealed various peaks related to magnetic nanoparticles, with additional peaks observed for the attachment of idarubicin, including planes at 2θ values of 400, 422, 511, and 440. These results confirmed the attachment of idarubicin to the surface of the nanoparticles. Thus, this study provides clear and supportive evidence for the current experimental results.

### 3.6. Anticancer potential of the MgO-IDA nanocomposite

#### 3.6.1. MTT assay analysis of MgO, IDA, and MgO-IDA nanocomposites

The metabolic activity of a cell, which is commonly employed as an indicator of cell viability, cytotoxicity, and cell proliferation, can be determined through the MTT assay. The metabolically active cells can reduce yellow-colored MTT reagents to purple-colored formazan crystals [56]. In the present study, an MTT assay was performed to investigate the in vitro cytotoxic nature of MgO nanoparticles, IDA, and MgO-
<figure><img src="image_9.png" /><figcaption>Fig. 7. Energy dispersive X-ray analysis of the MgO-IDA nanocomposite.</figcaption></figure>

<table><caption>**Table 1**

List of major and trace elements identified via EDX analysis.</caption><thead><tr><td>Element</td><td>Weight (%)</td><td>Atomic (%)</td><td>Net Int.</td></tr></thead><tbody><tr><td>C</td><td>35.71</td><td>49.5</td><td>1221.49</td></tr><tr><td>O</td><td>25.26</td><td>26.28</td><td>1659.15</td></tr><tr><td>Na</td><td>18.73</td><td>13.56</td><td>1985.8</td></tr><tr><td>Mg</td><td>5</td><td>3.42</td><td>618.98</td></tr><tr><td>N</td><td>0.42</td><td>0.22</td><td>45.82</td></tr><tr><td>H</td><td>13.83</td><td>6.5</td><td>1259.44</td></tr></tbody></table>

IDA nanocomposites against the MCF-7 breast cancer cell line. This test evaluated the ability of these nanoparticles and anticancer drugs to degrade cells.

Fig. 9 shows the excellent cytotoxic nature of the MgO-IDA nanocomposites in comparison with both the MgO nanoparticles and the idarubicin drug. The IC50 value, which represents the concentration required to reduce cell viability by up to 50 %, was significantly lower for the MgO-IDA nanocomposite (12 µg/mL). The lower the IC50 value of the nanocarrier drug is, the greater its efficiency in degrading cancerous cells. This reduced concentration revealed the enhanced

efficacy of the drug conjugated with nanoparticles in inducing cytotoxicity at very low dosages, highlighting its potential anticancer effects on breast cancer MCF-7 cell lines. These results support the use of MgO nanoparticles as promising carriers for the delivery of various drugs to treat breast cancer. They can also enhance the anticancer effects of chemotherapies [57].

Nigam et al., 2021, fabricated MgO nanoparticles in ZnO NPs to make MgO/ZnO nanocomposite and performed MTT assay to investigate the cytotoxic nature of MgO NPs and their nanocomposite against human liver and breast cancer i.e. HepG2 and MCF-7 cell lines, respectively. Their results highlighted the significant cytotoxic nature of MgO nanoparticles [58]. Hasan ULUSAL et al., 2022, synthesized glutaraldehyde coated MgO nanoparticles followed by immobilization of Idarubicin over the surface of NPs. This nanodrug was designed to investigate the effect of MgO NPs on the cytotoxic potential of anticancer drug i.e. Idarubicin. Their MTT assay on HL60 acute promyelocytic leukemia cells showed visible results exhibiting excellent cytotoxic activity of Idarubicin mobilized glutaraldehyde coated MgO NPs in comparison to free drug [55].

<figure><img src="image_10.png" /><figcaption>Fig. 8. X-ray diffraction analysis of the (a) MgO nanoparticles (b) MgO-IDA nanocomposite.</figcaption></figure>
<table><caption>**Table 2**
XRD pattern peak of the MgO-IDA nanoparticles.</caption><thead><tr><th>Peak No.</th><th>Angle (2θ)</th><th>Plane</th></tr></thead><tbody><tr><td>1</td><td>28°</td><td>111</td></tr><tr><td>2</td><td>33°</td><td>200</td></tr><tr><td>3</td><td>39°</td><td>220</td></tr><tr><td>4</td><td>48.5°</td><td>311</td></tr><tr><td>5</td><td>52°</td><td>422</td></tr><tr><td>6</td><td>57°</td><td>400</td></tr><tr><td>7</td><td>64°</td><td>440</td></tr></tbody></table>

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Cytotoxicity potential analysis of (mean ± SD) the MgO nanoparticles, IDA, and MgO-IDA nanocomposite after 24 h of incubation in the MCF-7 cell line of breast cancer (one-way ANOVA, *p* < 0.05).</figcaption></figure>

### 3.7. Cell migration analysis

Migration is a critical characteristic of cancer cells, allowing their growth, proliferation, and promotion of cellular metastasis. To investigate the effects of MgO nanoparticles, IDA, and MgO-IDA nanocomposites on cell migration activity, in vitro wound assays were performed on the MCF-7 breast cancer cell line. The cell metastasis behavior was determined at different time intervals, as shown in Fig. 10. The results revealed that cell metastasis was lower with the MgO-IDA nanocomposite than with the MgO nanoparticles or the IDA drug alone. The nanocomposite treatment reduced cell viability and metastasis by 30%, as shown in Table 3. The metastasis of cancer cells were determined after 48 h of treatment which showed activity bare MgO NPs and free IDA, there were 87.4% and 82.6% metastasis, respectively while for MgO-IDA nanocomposite these cells reduced 12.6% metastasis activity in comparison to free IDA drug. There is the significant reduction in the cancer cell malignancy while treating with drug nanocomposite. These results support the hypothesis that nanocarriers, when combined with drugs, more frequently reduce the migration of cancerous cells. A similar study was conducted to investigate the cell scratch distance after treatment with MgO nanoparticles, and the shortest distance was obtained with a high concentration of MgO nanoparticles [59].

### 3.8. Histochemical analysis of breast cancer tissues

The binding of folate-conjugated idarubicin with folic acid receptors on the surface of breast cancer tissue was assessed via histochemical analysis. The binding of idarubicin to folic acid receptors on the surface of breast cancer tissues was indicated by the appearance of red fluorescence in yellow and yellow color in red under a fluorescence microscope. However, idarubicin alone caused weak red fluorescence in the breast cancer tissue, whereas the MgO nanoparticles caused no

fluorescence. Fig. 11 shows the significant binding of folate ligands on the surface of the nanocarrier to the folic acid receptors of breast cancer tissues. Fig. 11a and b show the red color of the breast cancer tissues treated with the MgO-IDA nanocomposite in contrast to the yellow color, whereas Fig. 11c shows the same results with different color shades. Fig. 10d and e show breast cancer tissues treated with MgO nanoparticles and idarubicin, respectively. The MgO nanoparticles produced no fluorescence, whereas the idarubicin-treated slides presented slight fluorescence. These results highlight the potential of MgO-IDA nanocomposites for targeted drug delivery to breast cancer tissues. The folate ligand bound on the surface of the MgO-IDA nanocomposite targeted its receptors, which are specifically present in breast cancer tissues. In a previous study [60], histochemical analysis of dacarbazine-conjugated CdS quantum dots bound to folate receptors on the surface of HeLa cells revealed a red color under a fluorescence microscope. This is a promising tool for improved and more effective treatment of tumor cells.

### 3.9. Drug-releasing activity

Drug nanocarriers are designed to deliver drugs to target cancer cells. Idarubicin is conjugated to MgO nanoparticles via a covalent bond. The peptide bond between IDA and MgO nanoparticles is cleaved by the proteinase k enzyme inside lysosomes under lysosomal proteinase-conjugated particles at low pH (5.4) as lysosomal conditions [44]. The drug release efficiency was determined by measuring the UV-Vis absorbance at 540 nm and 482 nm, as shown in Fig. 12. After the initial 12 h, 40% of the drug was released in the presence of the enzyme, and 25% was released in the absence of the enzyme. After 96 h, 85% and 45% of the drug was released with and without the enzyme, respectively. Various nanoparticles are widely used as drug carriers in specific targeted drug delivery systems to treat cancer cells. Similarly, methotrexate-conjugated albumin-based nanoparticles loaded with curcumin have been designed for specific targeted delivery of multiple chemotherapeutic drugs and combination cancer drugs [61]. The results of that study showed that within 12 h intervals, proteinase k degrades the peptide bond and readily releases the drug from the nanocarrier at the site of melanoma cells.

### 3.10. Biosafety potential of the MgO-IDA nanocomposite

#### 3.10.1. Hemolysis assay

The assessment of the biocompatibility of drug nanocomposites with human blood components is necessary for biomedical applications. In this study, a hemolysis assay was performed to investigate the hemocompatibility of the MgO-IDA nanocomposite. According to the obtained results, the MgO-IDA nanoparticles have negligible impacts on the test concentrations, which confirms the minimal hemolysis effect of the human RBCs, as shown in Fig. 13. Hemolytic activity below 3.5 at high concentrations indicates the strong biocompatibility of human blood (RBC). These results support the nontoxic and nonhemolytic nature of MgO nanoparticles and provide safe and stable approaches for cancer therapy. This nonhemolytic nature of MgO nanoparticles was also confirmed by Velsanker et al. [62]. The authors synthesized MgO nanoparticles via a green synthesis approach involving *Oryza sativa* L. *indica* (black rice) extract. These authors demonstrated the excellent anticancer activity and nontoxic and nonhemolytic nature of MgO nanoparticles toward healthy cells and RBCs.

#### 3.11. Acute toxicity analysis of the MgO-IDA nanocomposite: in vivo studies

MgO nanoparticle inoculation at various dosages was nonlethal. However, groups 4 (350 mg kg⁻¹) and 5 (500 mg kg⁻¹) presented signs of lethargy, somnolence, occasional irritability, a ruffled coat, eye dilation, and decreased appetite, as shown in Table 4. Groups 1 (50 mg
<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Microscopic analysis at 10× magnification showing the migratory assay wound closure efficiency of the control sample and the samples treated with MgO nanoparticles, IDA, and the MgO-IDA nanocomposite. Scale bar, 100 µm.</figcaption></figure>

<table><caption>**Table 3**
Metastasis of cells at different time intervals in the migratory assay.</caption><thead><tr><th rowspan="2">Sample</th><th colspan="5">% Age Metastasis</th></tr><tr><th>0 h</th><th>6 h</th><th>24 h</th><th>48 h</th><th>control</th></tr></thead><tbody><tr><td>Control</td><td>85</td><td>90</td><td>95</td><td>100</td><td>0</td></tr><tr><td>MgO NPs</td><td>86.5</td><td>84.8</td><td>85.9</td><td>87.4</td><td>12.6</td></tr><tr><td>Free IDA</td><td>87.3</td><td>86.3</td><td>85</td><td>82.6</td><td>17.4</td></tr><tr><td>MgO-IDA nanocomposite</td><td>85.6</td><td>83.7</td><td>81.8</td><td>70</td><td>30</td></tr></tbody></table>

kg⁻¹), 2 (100 mg kg⁻¹), and 3 (250 mg kg⁻¹) demonstrated optimal safety, with no adverse effects noted. For the MgO nanoparticles, the optimal dosages were 100 and 250 mg kg⁻¹. Similarly, in the MgO-IDA nanocomposite inoculation group, all dosages were nonlethal. However, the mice in groups 9 (150 mg kg⁻¹) and 10 (200 mg kg⁻¹) were found to be somnolent, inactive, emaciated, and infrequently irritable (Table 5). The optimal dosages of idarubicin-conjugated MgO nanoparticles were 25, 50, and 100 mg kg⁻¹ for groups 6, 7, and 8, respectively. These results have also been compared with those of a previous study on the toxicity of MgO nanoparticles at different doses in mouse models. They employed various assays, i.e., hematology analysis, histological assay, and serum analysis, for the assessment of the toxicity level of MgO nanoparticles. These results showed that a high dose of MgO nanoparticles can increase the level of white blood cells and different enzymes. Histological analysis revealed some toxicity in liver tissues at high levels of MgO nanoparticles, whereas no toxicity was observed in

the kidney [63].

Overall, this study highlights the potential efficacy of MgO nanoparticles to deliver idarubicin specifically to its target sites with enhanced anticancer activity and reduced side effects. Further optimization, dosage control, and clinical trials are necessary to ensure the long-term safety of these nanocarriers in breast cancer treatment. This research provides a foundation for an innovative approach to designing higher-quality nanobased drug delivery carriers for the treatment of various cancerous disorders.

## 4. Conclusion

The stable, biocompatible, and targeted drug delivery system of MgO nanoparticles offers promising potential for treating breast cancer through conjugation with idarubicin, an anticancer drug. The combination of MgO nanoparticles with idarubicin significantly enhanced their therapeutic efficiency while reducing the off-target effects generally caused by conventional chemotherapeutic medications. The successful preparation of idarubicin and folic acid-conjugated BSA-coated MgO nanoparticles has been confirmed through various characterization methods, including UV-Vis spectroscopy, FT-IR, SEM-EDX, and XRD analysis. Furthermore, in vitro and in vivo studies revealed the potential of MgO nanoparticles to serve as highly stable, protective, and biocompatible drug-targeting agents to treat various tumor cells. Compared with free idarubicin, the results revealed the improved stability and anticancer activity of idarubicin conjugated with MgO nanoparticles. The results of the MTT assay and IC50 values from the cytotoxicity analysis revealed that the MgO nanoparticles significantly
<figure><img src="image_13.png" /><figcaption>**Fig. 11.** Fluorescence microscopy at 40× magnification showing the binding of folate with its receptors on the surface of breast cancer tissues. Scale bar, 200 µm. (a) and (b) Red color of the MgO-IDA-treated tissues is shaded in yellow. (c) Yellow color of the MgO-IDA-treated tissues is shaded in red. (d) No fluorescence in the MgO nanoparticles treated with idarubicin. (e) Slight fluorescence in the treated tissues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 12.** Idarubicin release efficiency in the presence and absence of proteinase K.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 13.** Hemolysis assay showing that flacons containing human RBCs with (a) deionized water caused hemolysis of RBCs, (b) PBS resulted in no hemolysis of RBCs, and (c) the MgO-IDA nanocomposite resulted in no hemolysis of RBCs.</figcaption></figure>

<table><caption>**Table 4**

Toxicity analysis of MgO nanoparticles in mice.</caption><thead><tr><th>MgO<br>nanoparticles<br>(mg kg<sup>-1</sup>)</th><th>Number of<br>mice/group</th><th>%age<br>Mortality</th><th>Signs of toxicity</th></tr></thead><tbody><tr><td>50</td><td>3</td><td>0</td><td>None</td></tr><tr><td>100</td><td>3</td><td>0</td><td>None</td></tr><tr><td>250</td><td>3</td><td>0</td><td>None</td></tr><tr><td>350</td><td>3</td><td>0</td><td>Hypo-activity/somnolence/<br>Moderate depression, gripping/<br>abnormal gait</td></tr><tr><td>500</td><td>3</td><td>0</td><td>Hypo-activity/Hyperventilation/<br>ruffle coat/decreased appetite and<br>reduced weight/lurching.</td></tr></tbody></table>

<table><caption>**Table 5**

Toxicity analysis of the MgO-IDA nanocomposite in mice.</caption><thead><tr><th>MgO<br>nanoparticle (mg<br>kg<sup>-1</sup>)</th><th>Number of<br>mice/group</th><th>%age<br>Mortality</th><th>Signs of toxicity</th></tr></thead><tbody><tr><td>25</td><td>3</td><td>0</td><td>None</td></tr><tr><td>50</td><td>3</td><td>0</td><td>None</td></tr><tr><td>100</td><td>3</td><td>0</td><td>None</td></tr><tr><td>150</td><td>3</td><td>0</td><td>Hypo-activity<br>Hypo-activity/<br>somnolence/Moderate<br>depression, gripping/<br>abnormal gait</td></tr><tr><td>200</td><td>3</td><td>0</td><td></td></tr></tbody></table>

inhibited the differentiation and proliferation of MCF-7 cancer cells through improved cellular uptake and continuous release profiles. This study highlighted the dual action of MgO nanoparticles as a promising platform not only for a specific drug delivery system but also for enhancing anticancer activity to treat breast cancer. This finding highlights the importance of nanotechnology in therapeutic and clinical applications.

## Cell lines

The human breast cancer cell line MCF-7 was obtained from ATCC. STR profiling and routine testing for mycoplasma contamination authenticated the cell line. The cells were cultured in high-glucose
DMEM supplemented with 10 % FBS and 1 % penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5 % CO2.

### CRediT authorship contribution statement

**Shoaib Shafique:** Resources, Investigation, Formal analysis. **Asma Irshad:** Writing – review & editing, Validation, Supervision, Formal analysis, Conceptualization. **Uzair Ishtiaq:** Writing – original draft, Validation, Resources, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Lotfi Mhamdi:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Sumaira Sharif:** Methodology, Investigation. **Rabbia Jawad:** Software, Data curation. **Patrick Martin:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. **Naceur M’Hamdi:** Validation, Resources, Methodology. **Saad Arshad:** Validation, Resources, Investigation. **Bochra Bejaoui:** Investigation, Formal analysis, Data curation.

### Consent for publication

We declare that this work is original and not under consideration by any other journal or published in any other journal.

### Ethics approval and consent to participate

The study involving human blood samples was conducted in accordance with the Declaration of Helsinki. Ethical approvals were obtained from the University of the Punjab Ethical Committee (IBB/24/1082), and Artois University (92C24/31). Informed consent was obtained from all volunteers.

### Funding

There was no funding available for this study.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

All the data used for this study are presented in this manuscript.

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