Functionalized magnetic nanoparticles orchestrate PI3K/AKT signaling to promote apoptosis in breast cancer cells

Abstract

Herein, we report the molecular mechanism by which bare (BMNPs) and citrate-functionalized magnetic nanoparticles (CMNPs) target apoptosis by modulating the PI3K/AKT signaling pathway in breast cancer cells. To achieve this, BMNPs and CMNPs were prepared via co-precipitation and characterized using high-throughput techniques. Furthermore, the as-prepared nanomaterials were evaluated for their anticancer efficacy in modulating apoptosis in breast cancer cells (MCF-7 and MDA-MB-231). The following observations were made: (i) citrate functionalization provided CMNPs with a reactive carboxyl-rich surface, reduced aggregation and improved the zeta potential value (−20.8 mV); (ii) CMNPs demonstrated greater selectivity against MCF-7 and MDA-MB-231 than BMNPs, as revealed by the MTT assay and clonogenic assay; (iii) CMNPs generated high intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential in breast cancer cells; (iv) CMNPs demonstrated greater cellular uptake, G1-phase cell-cycle arrest, and anti-migratory activity than BMNPs; and (v) mechanistic validation confirmed that CMNPs suppressed the oncogenic PI3K/AKT signaling pathway and activated the intrinsic mitochondrial apoptotic cascade in breast cancer cells (MCF-7 and MDA-MB-231). These findings reveal the influence of surface chemistry on MNP–biological interactions, offering essential mechanistic insights into nano-oncology and paving the way for the development of innovative nanoplatforms for targeted cancer therapy.

Herein, we report the molecular mechanism by which bare (BMNPs) and citrate-functionalized magnetic nanoparticles (CMNPs) target apoptosis by modulating the PI3K/AKT signaling pathway in breast cancer cells.

1. Introduction

According to the National Centre for Health Statistics (2022), the morbidity and mortality rates of cancer, a non-communicable disease, are escalating daily.¹ Despite significant advancements in the search for anticancer drugs, barriers persist in translating treatments from preclinical studies into clinical practice.^2,3 These challenges may stem from the substantial developmental costs and the difficulties in targeting cancer cells without adversely affecting normal cells. Hence, there is a need for the development of novel drug delivery systems, which can address these restrictions.⁴

Thus far, several nanomaterials, including carbon nanotubes, quantum dots, liposomes, dendrimers, mesoporous silica and metallic nanoparticles, have shown significant progress in the biomedical sciences.^5,6 In particular, magnetic nanoparticles (MNPs) composed of iron oxide have transformed specific domains such as diagnosis, bioimaging, targeted drug delivery, and cell therapy.^7,8 For instance, MNPs have been accurately directed to particular tissues or organs utilizing an external magnetic field for targeted drug administration.⁹ To ensure biocompatibility, MNPs can be coated or surface-functionalized with bio-inert chemical agents to mitigate side effects, rendering them appropriate for in vivo applications. MNPs can also serve as MRI contrast agents for the early identification of sickness or inflammation within living systems.¹⁰ In this way, MNPs bridge nanotechnology and medicine by facilitating diagnostic and therapeutic applications that are essential for next-generation personalized medicine.¹¹

Among MNPs, maghemite (γ-Fe₂O₃) nanoparticles (BMNPs) have been extensively explored for their intrinsic anticancer properties.¹² In most cases, BMNPs are synthesized via the co-precipitation, thermal decomposition and laser target evaporation (LTE) methods.¹³ For example, BMNPs synthesized via co-precipitation showcased minimal toxicity below 100 µg mL⁻¹, confirming their safety for direct tumor injection.^14,15 Likewise, the LTE-mediated preparation of BMNPs resulted in the generation of reactive oxygen species (ROS) at elevated concentrations, contributing to cytotoxicity in human dermal fibroblast cells under an external magnetic field.^16,17 In the same way, BMNPs displayed strong γ-ray absorption, suggesting their potential as sensitizers in radiotherapy.¹⁸ In addition, BMNPs induced cell death in A549 and MCF-7 cells via generating ROS and DNA fragmentation.^19,20 Despite numerous studies, fundamental limitations in colloidal stability, biocompatibility, tumor targeting specificity, pharmacokinetics and therapeutic efficacy of BMNPs need to be addressed.²¹

In order to overcome these limitations, considerable efforts have focused on the surface functionalization of BMNPs, which has shown potential to translate these nanomaterials from bench to bedside. For example, surface coating with polyethylene glycol, silica or small molecules provides both electrostatic and steric stabilization.^22,23 DMSA-coated maghemite nanoparticles showed no cytotoxic effects on macrophages at concentrations up to 1 × 10¹⁵ particles per mL, whereas uncoated particles induced dose-dependent cell death.²⁴ Similarly, PEG-coated iron-oxide nanoparticles obstructed the reduction of cytochrome c (a key mitochondrial protein), while bare nanoparticles caused protein denaturation.²⁵ Similarly, silica-coated maghemite nanoparticles exhibited >80% cell viability in fibroblast assays even after 72 hours of exposure, confirming their excellent biocompatibility as drug delivery platforms.²⁶

Beyond improving biocompatibility, surface functionalization of BMNPs has also been explored for the development of multifunctional therapeutic nanocarriers. Adding more insights, lactoferrin-coated maghemite nanoparticles loaded with doxorubicin showed superior antimetastatic activity in breast cancer models.²⁷ In the same way, a rhodium(ii) citrate-loaded system demonstrated profound antitumour effects against metastatic breast cancer, with prolonged survival and minimal system toxicity.²⁸ A tri-stimuli-responsive system (maghemite/PLGA/chitosan) combination exhibited 4.7-fold faster drug release under tumor-like conditions and 1.6-fold lower half-maximum inhibitory values than free cisplatin.²⁹ The development of folate-modified, albumin-functionalized iron oxide nanoparticles demonstrated selective accumulation in cancer cells with increased folate receptor expression, enabling targeted photodynamic therapy with enhanced efficacy compared to free photosensitizers.³⁰ Novel iron oxide nanoparticles coated with gallic acid and polyacrylic acid (IONP-GA/PAA) demonstrated intrinsic cytotoxic activity through efficient ferroptosis induction in glioblastoma, neuroblastoma, and fibrosarcoma cells.³¹ Although several studies demonstrate the versatility of functionalized magnetic nanoplatforms, the majority rely on therapeutic cargo such as drugs, ligands and multimodal formulations, which makes it difficult to distinguish the intrinsic biological contributions of surface functionalization.

Meanwhile, capping BMNPs with citrate provides crucial surface stabilization while facilitating selective cancer cell targeting due to their negatively charged carboxylate groups.^32,33 Previous research studies document that BMNPs and functionalized iron oxide nanoparticles represent highly promising anticancer platforms with multiple therapeutic mechanisms, including targeted drug delivery, hyperthermia, immunomodulation, and ferroptosis induction.^34,35 It has also been reported that the functionalization of BMNPs with citrate demonstrates enhanced cellular uptake.³⁶ However, its direct influence on the intrinsic molecular mechanism regulating apoptosis in breast cancer is poorly understood. Despite their potential as drug carriers and imaging agents,³⁷ only a few studies have systematically compared the intrinsic biological activity of bare and citrate-functionalized nanoparticles in the absence of therapeutic cargo. Moreover, the influence of citrate functionalization on ROS-mediated mitochondrial dysfunction and PI3K/AKT signaling in distinct breast cancer subtypes remains poorly understood.

Keeping this in mind, the present study aims to elucidate the intrinsic molecular effects of citrate-functionalized BMNPs in targeting the PI3K/AKT signaling pathway in breast cancer cells (MCF-7 and MDA-MB-231). We hypothesize that functionalized BMNPs orchestrate apoptosis by inducing mitochondria dysfunction, manifested by a loss of mitochondrial membrane potential and increased ROS production, leading to the suppression of the PI3K/AKT signaling pathway. To the best of our knowledge, this is the first study to systematically compare bare maghemite nanoparticles (BMNPs) and citrate-functionalized maghemite nanoparticles (CMNPs) without a therapeutic cargo by integrating physicochemical characterization with mechanistic investigations of ROS generation, mitochondrial dysfunction, apoptosis, and PI3K/AKT signaling. Altogether, the findings from this study provide new insights into the rational design of surface-engineered MNPs for breast cancer therapy and support the development of multifunctional theranostic platforms.

2. Materials and methods

2.1. Materials

All chemicals and reagents used in the study for the preparation of nanomaterials were procured from Sigma-Aldrich Pvt. Ltd, Bangalore, India. HiMedia Pvt. Ltd, Bangalore, provided the media and chemicals required for cell culture studies. Primers used in the study were designed by EuroFins Genomics India Pvt. Ltd, Bangalore, India. Primary and secondary antibodies were obtained from Cell Signaling Technologies, USA.

2.2. Synthesis of magnetite (Fe₃O₄) nanoparticles

In the present study, iron oxide nanoparticles were synthesized using a co-precipitation method.¹⁵ An aqueous solution containing FeCl₂ and FeCl₃ in a 2 : 1 molar ratio, acidified with 0.4 M HCl, was rapidly added to 2.5 M ammonium hydroxide under vigorous stirring (1000 rpm) at room temperature. This process resulted in the immediate formation of a black precipitate corresponding to magnetite (Fe₃O₄). The magnetite precipitate was magnetically separated from the reaction mixture and washed repeatedly with distilled water until a neutral pH was achieved. Subsequently, the washed magnetite nanoparticles were left to stand undisturbed in ambient air to facilitate atmospheric oxidation, which converted the magnetite phase to maghemite (γ-Fe₂O₃). The resulting bare maghemite nanoparticles (BMNPs) were then washed multiple times with distilled water to remove any residual impurities and finally resuspended in deionized water for further characterization and subsequent applications.

2.3. Conjugation studies

Further, trisodium citrate was conjugated to the surface of BMNPs at a 1 : 10 BMNPs-to-trisodium citrate ratio using a post-synthesis functionalization approach.³⁸ BMNPs were dispersed in an aqueous solution, and trisodium citrate dihydrate (Na₃C₆H₅O₇·2H₂O) was added under controlled pH conditions (typically pH 8–10). The mixture was stirred at an elevated temperature (60–80 °C) for 1–2 h to facilitate ligand exchange and surface coordination. The final product obtained was centrifuged thrice at 12000 rpm, and the resulting pellet was dried in a hot air oven before further use.

2.4. High-throughput characterization

The as-prepared BMNPs and CMNPs were characterized using advanced analytical instruments to elucidate their properties. To identify the surface functional groups and confirm the presence of chemical bonds, Fourier-transform infrared (FTIR) spectroscopy was performed using a Thermo Scientific™ Nicolet™ iS™5 FT-IR spectrometer, USA. Complementary vibrations and phase verification were evaluated using micro-Raman spectroscopy (Seiki, Japan). The crystalline structure was examined by X-ray diffraction (XRD) employing an X-Pert Pro diffractometer (PANalytical, UK). Field-emission scanning electron microscopy (FE-SEM, Carl Zeiss Model: EVO19 SEM) coupled with EDX was used to obtain morphological features and elemental composition. High-resolution transmission electron microscopy (HR-TEM, JEOL JEM-2100 PLUS, Japan) and selected-area electron diffraction (SAED) further characterized the particle size, lattice fringes, and crystallinity. A vibrating sample magnetometer (Cryogenic, UK) was used for magnetization measurements to evaluate the magnetic behaviour and confirm superparamagnetic properties. Dynamic light scattering (DLS) measurements were performed using a Zetasizer (Malvern Instruments, UK) to ascertain the average hydrodynamic size distribution and surface charge. Thermogravimetric analysis was performed using a simultaneous TGA/DSC system (ambient temperature to 1500 °C).

2.5. Cell lines used

Human breast cancer cell lines (MCF-7 and MDA-MB-231) and the non-cancerous human embryonic kidney cell line (HEK-293) used in the study were procured from the National Centre of Cell Science (NCCS), Pune. The obtained cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antimycotic solution. Using a humidified incubator, the cells obtained were maintained at 37 °C with 5% CO₂. The culture medium was refreshed every 2–3 days to ensure optimal growth and viability. All experiments were performed with cells between passages 5 and 15. No live animals or human participants were involved in this study. The cell lines used are established and do not require ethical approval for use.

2.6. MTT assay

An MTT assay was performed to assess the cell viability of BMNPs and CMNPs against breast cancer cell lines (MCF-7 and MDA-MB-231) and the non-cancerous human embryonic cell line (HEK-293) at different concentrations (0–200 µg mL⁻¹), based on the methodology adopted by Mosmann in 1983.³⁹ In this study, the MTT dye is reduced in the presence of mitochondrial dehydrogenase enzymes in metabolically active cells, resulting in the formation of insoluble purple formazan crystals. Dimethyl sulfoxide (DMSO) solubilized the as-formed formazan crystals to produce a colored solution. The microtitre plate reader measured the absorbance of the colored solution at 595 nm to ascertain the number of viable cells.

2.7. Clonogenic assay

A clonogenic assay was performed to measure single-cell reproductive viability by assessing colony growth upon treatment with the IC₅₀ concentrations of CMNPs and BMNPs against breast cancer cells (MCF-7 and MDA-MB-231), which was based on a methodology reported previously.⁴⁰ At the end of the experiments, colonies were fixed using ethanol and stained with crystal violet to visualize the colonies. The excess dye was removed using distilled water, and the colonies were further counted and recorded using a stereomicroscope (Debro-DZ 55, India).

2.8. Fluorescence microscopic analysis

In our study, fluorescence-based stains, such as dual stains (acridine orange (AO) and ethidium bromide (EtBr)),⁴¹ Hoechst 33342,²⁶ DCFH-DA,⁴² and Rhodamine 123,⁴³ were used to study apoptosis, nuclear damage, oxidative stress, and mitochondrial health, respectively. Subsequent to treatment with BMNPs and CMNPs at their IC₅₀, the breast cancer cells were gently washed thrice with phosphate buffered saline (PBS) to remove residual nanoparticles. Appropriate fluorescent dyes AO (1 µL)/EtBr (1 µL) were mixed and incubated for 15 min; Hoechst 33342 (2 µL, 15 min); DCFH-DA (2 µL, 30 min); and Rhodamine 123 (2 µL, 30 min), under optimized staining conditions with all incubations performed at 37 °C in the dark. After incubation, excess dye was removed by washing the cells thrice with PBS. A fluorescence microscope (Accu-Scope, EXO-310) was used to acquire micrographs at 20× magnification. We also performed fluorescence-based staining with AO/EtBr and Hoechst 33342 for HEK-293 cells upon treatment with BMNPs and CMNPs (150 µg mL⁻¹), and the cells were imaged under similar experimental conditions.

2.9. Cell migration assay

A scratch assay was performed to study the migration of cells based on the methodology reported elsewhere.⁴⁴ Upon treatment with the IC₅₀ concentrations of BMNPs and CMNPs, the migration of breast cancer cells was imaged at pre-determined intervals (0–48 h) to assess the closure of the wound using an inverted phase-contrast microscope (Accu-Scope, EXI-310, USA).

2.10. Cellular uptake by Prussian blue staining

The cellular uptake of BMNPs (50 µg mL⁻¹) and CMNPs (50 µg mL⁻¹) was ascertained using Prussian blue staining.⁴⁵ In our study, treated breast cancer cells were stained with Prussian blue for 30 min. After incubation, the samples were rinsed thoroughly with PBS to remove excess stain and were visualized under a phase-contrast microscope at 20× magnification to ascertain the presence of MNPs within the breast cancer cells.

2.11. Cell cycle analysis

To study the impact of BMNPs and CMNPs on the progression of the cell cycle, propidium iodide (PI) staining combined with flow cytometry was used.⁴⁰ Breast cancer cells, after treatment with BMNPs and CMNPs, were trypsinized, washed (with PBS), fixed (70% ethanol), and stored overnight (at −20 °C). Subsequently, the pre-fixed cells were incubated with RNaseA at 37 °C for 1 hour and stained with PI for 15 min at room temperature. In the end, a FACS Fortessa X-20 was used to determine the cell cycle phase, and the results were analyzed by the FlowJo software.

2.12. Annexin V-FITC/propidium iodide (PI) apoptosis assay

The progression of apoptosis in breast cancer cells (MCF-7 and MDA-MB-231) was evaluated using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit according to the manufacturer's instructions.⁴⁶ Briefly, cells treated with the IC₅₀ concentrations of BMNPs and CMNPs for 24 h were used in the study. Untreated cells served as controls. Subsequent to treatment, the cells were trypsinized, washed twice with ice-cold PBS and resuspended in 100 µL of binding buffer. Later, 5 µL of Annexin V-FITC and 5 µL PI were added to each sample, followed by gentle mixing and incubation for 15 min at room temperature in the dark. Flow cytometric analysis was performed using a Sysmex Partec GmbH Cyflow Cube 6 cytometer (Japan), and the obtained data were analyzed using the FlowJo software.

2.13. Gene expression

Total RNA was isolated from breast cancer cells treated with the IC₅₀ concentrations of BMNPs and CMNPs using TRIzol reagent and quantified using a fluorometer (Quantus™ Fluorometer, Promega, USA). The quantified total RNA was used for cDNA synthesis (PrimeScript 1st Strand cDNA Synthesis Kit one-step RT-PCR, Takara, Japan).⁴⁷ A qRT-PCR study was then performed to determine the expression pattern of genes involved in the induction of apoptosis, as shown in SI Table 1.

2.14. Western blotting analysis

Proteins from MCF-7 and MDA-MB-231 were extracted using RIPA lysis buffer after treatment with the IC₅₀ concentrations of BMNPs and CMNPs. About 50 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The obtained gel was electro-blotted onto a nitrocellulose membrane, followed by blocking using non-fat dry milk for 1 h at 37 °C. The blocked membrane was incubated with primary antibodies (PI3K, p-AKT, Bcl-2, caspase-3, and GAPDH) overnight at 4 °C. After incubation, the membrane was washed thrice with tris-buffered saline and further incubated with secondary antibodies for 2 h at room temperature. In the end, the blots were developed using a chromogenic solution [5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT)] and visualized using a bioimaging system (ImageQuant, LAS-500 GE, USA). GAPDH was used as an internal control.

2.15. Statistical analysis

In our study, statistical analysis was performed using GraphPad Prism V.9.3 statistical software, with data presented as mean ± standard error (SE) of three independent experiments. For comparison (control, BMNPs, and CMNPs), we used one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparisons post hoc test. A p value <0.05 was considered statistically significant. Statistical significance was denoted as follows: p < 0.05, *p < 0.01, **p < 0.001, ***p < 0.0001 and ns – non-significant.

3. Results and discussion

In the present study, maghemite nanoparticles (BMNPs) were synthesized using a co-precipitation approach, where FeCl₂ and FeCl₃ served as the precursor salts, leading to the formation of a black precipitate, magnetite (Fe₃O₄), which subsequently oxidized to form maghemite (γ-Fe₂O₃) under ambient conditions.^48,49 Surface modification with trisodium citrate effectively stabilized the maghemite nanoparticles (CMNPs), significantly enhancing their dispersibility and biocompatibility, while providing reactive surface chemistry that may expand their potential for biomedical applications.^15,20 To understand the functional moieties present on the surfaces of BMNPs and CMNPs, we performed FTIR spectroscopy analysis, as shown in SI Fig. 1. For BMNPs, the presence of weak peaks at 3417 cm⁻¹ and 1625 cm⁻¹ suggests the presence of hydroxyl groups, originating from O–H stretching vibrations.⁵⁰ A prominent peak at 576 cm⁻¹ confirms the spinel structure of Fe–O stretching vibrations.^51,52 In contrast, the FTIR spectrum of CMNPs reveals distinct carboxylate-associated peaks at 1610 cm⁻¹ (C O asymmetric stretching) and 1403 cm⁻¹ (C–O symmetric stretching) following functionalization with trisodium citrate.⁵³ The retention of the Fe–O vibration at 572 cm⁻¹ indicates the structural integrity of the maghemite core, which remains unaffected during the functionalization process.⁵⁴

The Raman spectrum of BMNPs further exhibited three prominent peaks at 200 cm⁻¹ (A_1g), 250 cm⁻¹ (T_2g), and 400 cm⁻¹ (E_g), which correspond to Fe–O lattice vibrations, Fe–O bonding, and nanocrystalline Fe₂O₃, respectively (SI Fig. 2a). These results aligns well with findings in the literature related to maghemite Fe₂O₃.⁵⁵ A strong band centered at 1350 cm⁻¹ is linked to the D-band of disordered carbon, which results from surface defects introduced during nanoparticle synthesis.⁵⁶ The absence of a G-band (1580 cm⁻¹) indicates a minimal carbon content.⁵⁷ In contrast, CMNPs exhibited broader and less intense signals below 700 cm⁻¹, suggesting Fe–O vibrations compared to those of BMNPs (SI Fig. 2b). Notably, a broad band in the 1300–1500 cm⁻¹ region is attributed to citrate capping, stemming from the binding of Fe³⁺ sites via carboxyl groups.⁵⁸ The increased intensity in this region is characteristic of C–H and C–C bending vibrations from citrate molecules, confirming their role as capping agents.⁵⁹

SI Fig. 3 displays the XRD patterns of the as-prepared BMNPs and CMNPs, which exhibit characteristic diffraction peaks at 2θ values corresponding to the (220), (311), (400), (422), (511), and (440) crystallographic planes, confirming the presence of the cubic spinel structure of maghemite (γ-Fe₂O₃).⁶⁰ No additional diffraction peaks were observed after citrate functionalization, indicating that the crystalline phase of MNPs remained unchanged. In our study, the slight decrease in peak intensity for CMNPs can be linked to the presence of an amorphous citrate layer on the surface of CMNPs rather than to amorphization of the maghemite core.^61,62 Further, the average apparent crystallite size was estimated using Scherrer's equation from the most intense (311) diffraction peak and was found to be 23 and 32 nm for BMNPs and CMNPs, respectively. It is also noted from the study that the apparent crystallite size observed for CMNPs is attributed to reduced peak broadening after citrate functionalization rather than an increase in the crystalline maghemite core size.

In addition, the XPS spectra of BMNPs show Fe 2p peaks at 711 and 725 eV with a distinct satellite near 719 eV, confirming a γ-Fe₂O₃ core with Fe predominantly in the Fe³⁺ state and only minor Fe²⁺ (SI Fig. 4a–c). The O 1s signal is dominated by lattice O²⁻ (Fe–O) around 530 eV with a smaller OH/H₂O contribution at a higher binding energy, indicating a mainly oxide surface with limited hydroxylation. After citrate stabilization (CMNPs), the Fe 2p features of maghemite are preserved, demonstrating that the organic modification does not alter the iron oxidation state or core phase (Fig. 1a–d). In contrast, the C 1s and O 1s regions change markedly: new or intensified components at 288 eV (C–O, COO⁻/COOH) and 532 eV (C–O/C O) arise from the carboxylate groups of surface-bound citrate, and the C 1s atomic percentage increases to ∼36%, confirming dense organic coverage. Based on the observation, XPS shows that BMNPs are phase-pure maghemite with a relatively inorganic surface, whereas CMNPs comprise the same magnetic core wrapped by a carboxylate-rich citrate shell that enhances hydrophilicity, colloidal stability, and offers reactive sites for further bioconjugation.

Fig. 1. XPS spectra of CMNPs. (a) C 1s region fitted into C–C/C–H, C–O and COO⁻/COOH components, evidencing the presence of citrate ligands on the nanoparticle surface. (b) O 1s spectra showing contributions from Fe–O/C O and C–O/H–O species associated with the citrate shell. (c) Fe 2p spectra retaining the Fe³⁺ features of γ-Fe₂O₃, indicating preservation of the magnetic core after functionalization. (d) Survey spectrum highlighting increased carbon content relative to BMNPs due to citrate coating while maintaining strong Fe and O signals from the underlying iron oxide core.

The morphological characteristics of BMNPs and CMNPs were analyzed using FE-SEM in conjunction with EDX analysis. Fig. 2a–c illustrate that BMNPs are spherical, measuring between 20 and 30 nm, and show signs of agglomeration.^63,64 In contrast, after conjugation with trisodium citrate, these particles displayed a more spherical shape and an increased size, ranging from 30 to 35 nm, with no signs of agglomeration (Fig. 2d–f). The particle size measured by SEM corresponds well with the calculated values obtained from the XRD analysis. The EDX spectrum of BMNPs shows intense peaks for Fe (56%) and O (44%), while that of CMNPs show the existence of C, Fe, and O peaks, designating the successful conjugation of citrate ions onto the CMNP surface (SI Fig. 5a and b).

Fig. 2. Scanning electron micrographs at different magnifications for BMNPs (a–c) and CMNPs (d–f).

To further validate these findings, HR-TEM analysis was performed to evaluate the size and shape of BMNPs (SI Fig. 6) and CMNPs (Fig. 3). The results show that CMNPs maintain a distinct spherical shape with mild polydispersity (Fig. 3a–e). It has been reported that the conjugation of trisodium citrate exerts an electrostatic repulsive force on CMNPs, preventing their agglomeration.³⁸ Previous studies suggest that iron oxide nanoparticles produced via co-precipitation are highly agglomerated, with typical diameters ranging from 20 to 30 nm, primarily due to elevated temperatures.⁶⁴ The average size range of CMNPs was 25 to 35 nm, closely matching the results from the SEM and XRD analyses. Moreover, the SAED patterns confirm the spinel-phase characteristics of Fe₂O₃. Fig. 3f depicts the SAED pattern, displaying diffraction spots indicative of the highly crystalline lattice fringes of CMNPs (Fig. 3a–c).

Fig. 3. High-resolution transmission electron micrographs at different magnifications for CMNPs (a–e) and SAED pattern (f).

The magnetic properties of BMNPs and CMNPS were evaluated using vibrating sample magnetometry (VSM) at room temperature (Fig. 4). In our study, the saturation magnetization (M_s) of BMNPs was determined to be 46.75 emu g⁻¹, whereas CMNPs exhibited a higher M_s value of 65.05 emu g⁻¹, suggesting that citrate functionalization enhanced the magnetic response of the nanoparticles without altering their magnetic core. Moreover, citrate functionalization enhanced the colloidal stability and reduced magnetic dipole–dipole interactions, which tend to minimize aggregation and thereby facilitate efficient magnetic alignment under an applied magnetic field.⁶⁵ It is noticed from our study that CMNPs exhibited M–H hysteresis loops with very low coercivity (H_c = ∼61.7 Oe) and a low remanence ratio (M_r/M_s = 0.074), demonstrating soft magnetic behaviour with superparamagnetic-like characteristics (SI Table 2). Although the narrow hysteresis loops suggest excellent magnetic reversibility, the non-zero coercivity and remanence indicate that the nanoparticles cannot be conclusively classified as superparamagnetic based solely on room-temperature VSM measurements.⁶⁶ On the other hand, the enhanced saturation magnetization together with the soft magnetic characteristics of CMNPs makes them promising candidates for magnetically guided drug delivery, magnetic separation, and magnetic hyperthermia applications.^67–69

Fig. 4. Magnetic behavior of BMNPs and CMNPs obtained using a vibrating sample magnetometer (VSM).

Dynamic light scattering (DLS) analysis was performed to evaluate the hydrodynamic size distribution of the as-prepared BMNPs and CMNPS in aqueous suspension. As shown in SI Fig. 7a and b, CMNPs exhibited a relatively smaller hydrodynamic diameter (156.3 nm) compared to that of BMNPs (293.5 nm). Altogether, a slight decrease in the polydispersity index (PDI) was noted for CMNPs (0.442) compared with BMNPs (0.497), suggesting a modest improvement in particle size homogeneity following citrate functionalization.⁷⁰ The reduction in hydrodynamic diameter following citrate conjugation suggests that surface functionalization improved nanoparticle dispersion and reduced particle agglomeration, as seen in Fig. 3.

Furthermore, the surface charge values of MNPs used in the study were determined by zeta potential measurements, as presented in SI Fig. 7c and d. In our study, BMNPs exhibited a zeta potential value of −15.8 mV, whereas CMNPs displayed a more negative zeta potential value of −20.8 mV.⁷¹ The increase in the magnitude of the zeta potential for CMNPs can be attributed to the presence of carbonyl (–COO⁻) groups of citrate molecules anchored onto the surface of CMNPs, as confirmed by FTIR and XPS analyses. As a result, an increase in electrostatic repulsion between adjacent CMNPs is noticed, thereby reducing particle aggregation and improving colloidal stability in aqueous media.

To ascertain the thermal stability and verify the successful surface functionalization of citrate onto the surface of BMNPs, thermogravimetric analysis was performed. As shown in SI Fig. 8a and b, both BMNPs and CMNPs exhibited a multistep weight-loss profile over the investigated temperature range.⁷² At 150 °C, an initial weight loss is observed for both samples, attributed to the evaporation of physically adsorbed water molecules and solvents on the surface. A second stage of weight loss occurs approximately between 200 °C and 450 °C, inferring the thermal decomposition of surface-bound organic components. At this stage, CMNPs exhibited a greater weight loss than BMNPs, indicating the thermal degradation of citrate-derived organic moieties chemically grafted onto the surface of BMNPs. Above 450 °C, minimal weight loss is noticed, inferring that the inorganic magnetic core remained thermally stable at higher temperatures. Based on the observations, TGA studies confirm the successful conjugation of citrate onto the surface of MNPs.

The in vitro cytotoxic potential of BMNPs and CMNPs was evaluated against breast cancer cells (MCF-7 and MDA-MB-231) and normal cells (HEK-293) using the MTT assay. As shown in Fig. 5, the ability of BMNPs and CMNPs to inhibit cell proliferation was concentration-dependent, highlighted by the half-maximum inhibitory (IC₅₀) values reported in SI Table 3. The study also noted that CMNPs exhibited significantly pronounced cytotoxicity in breast cancer cells than BMNPs. Escamilla-Rivera et al. reported that cytotoxic iron oxide nanoparticles conjugated with PEG exhibited a similar effect against THP-1 macrophages after 48 hours of treatment.⁷³ Likewise, Schweiger et al. reported polymer-capped iron oxide magnetic nanoparticles demonstrating cytotoxic effects against A549 epithelial adenocarcinoma cells.⁷⁴

Fig. 5. BMNPs and CMNPs induce potent cytotoxic and anticlonogenic effects in breast cancer cells. Dose-dependent cytotoxicity of BMNPs and CMNPs in (i) MCF-7 and (ii) MDA-MB-231 cells assessed by MTT assay after 24 h of exposure. (iii–v) Representative clonogenic survival of MCF-7 cells upon treatment with (iii) untreated control, (iv) BMNPs and (v) CMNPs, and (vi) corresponding quantitative analysis of colony numbers. (vii–ix) Clonogenic survival of MDA-MB-231 upon treatment with (vii) untreated control, (viii) BMNPs, and (ix) CMNPs, and (x) corresponding colony quantification. Data are presented as mean ± SD from three independent experiments.

Taking this into consideration, this study suggests that trisodium citrate enhances the cytotoxic efficacy of MNPs in targeting breast cancer cells.⁷⁵ Meanwhile, the cell viability of BMNPs and CMNPs in normal, non-cancerous cells is shown in SI Fig. 9. The results from the study indicate that an increasing drug concentration was non-toxic to normal cells (HEK-293), highlighting the biocompatibility. In view of this, the selectivity index was calculated, which is shown in SI Table 4. Furthermore, a clonogenic assay was conducted in the presence of BMNPs and CMNPs to ascertain the long-term cell proliferation in breast cancer cells (Fig. 5). The results from the study exemplify that the IC₅₀ concentration of CMNPs significantly reduced the colony numbers of breast cancer cells more than BMNPs. Based on the above information, it is clear that MNPs possess a cytostatic effect on breast cancer cells by impeding their colony-forming ability.^76,77

In light of the above evidence, we meticulously examined the role of fluorescent probes in assessing the progression of apoptosis. Initially, a dual fluorescent probe (AO/EtBr) was employed to study the response induced by MNPs in breast cancer cells (Fig. 6A). When visualized under a fluorescence microscope, AO stains both viable and non-viable cells green.⁷⁸ In contrast, EtBr stains non-viable cells red due to their compromised cytoplasmic membrane integrity.⁷⁹ In our study, the cells exposed to the IC₅₀ concentration of BMNPs and CMNPs exhibited characteristic features of apoptosis. The study results indicated that the CMNP-treated cells displayed more apoptotic cells than the BMNP-treated cells. This may be due to the rapid internalization of CMNPs, leading to superior cytotoxicity compared to BMNPs.⁸⁰

Fig. 6. Dual and nuclear fluorescent staining of BMNPs and CMNPs that induce apoptotic cell death in breast cancer cells. (A) Live/dead staining (acridine orange/ethidium bromide) of MCF-7 and MDA-MB-231 cells treated with BMNPs or CMNPs at their IC₅₀ concentrations for 24 h. Live cells: fluorescence green and dead cells: red. Untreated cells show normal morphology for MCF-7 (i) and MDA-MB-231 (iv) cells, whereas BMNP-treated MCF-7 (ii) and MDA-MB-231 (v) cells and CMNP-treated MCF-7 (iii) and MDA-MB-231 (vi) cells exhibit increased cell death. (vii) Quantification of apoptotic, pyknotic, and non-apoptotic cells. (B) Hoechst 33342 staining of MCF-7 and MDA-MB-231 cells. Untreated cells show no pyknotic nuclei for MCF-7 (i) and MDA-MB-231 (iv) cells, whereas treatment with BMNPs shows signs of pyknotic nuclei for MCF-7 (ii) and MDA-MB-231 (v) cells, and CMNP-treated cells showed a slight increase in pyknotic nuclei ratio in both MCF-7 (iii) and MDA-MB-231 (vi) cells. (vii) Corresponding quantification of pyknotic nuclei. Data are presented as mean ± SD (n = 3).

Secondly, nuclear alterations involving Hoechst 33342 staining were studied, as it preferentially binds AT-rich DNA regions in cells with compromised membrane integrity (Fig. 6B).⁸¹ In our study, treatment with BMNPs and CMNPs showed prominent nuclear fragmentation, with blue fluorescence upon Hoechst 33342 staining, in contrast to the control. Crucial apoptotic features like nuclear fragmentation and chromatin condensation were noticed in breast cancer cells.⁸² Notably, CMNPs exhibited a higher frequency of fragmented nuclei, indicating an enhanced apoptosis rate due to nuclear damage compared to BMNPs.⁸³ A study presenting superparamagnetic iron oxide nanoparticles (SPIONs) showed nuclear damage in murine macrophage (J774) cells, consistent with our research.⁸⁴ In our study, neither BMNPs nor CMNPs induced any apoptotic events or observable morphological changes in HEK-293 cells, as evidenced in SI Fig. 10.

Thirdly, the generation of reactive oxygen species (ROS) induced by BMNPs and CMNPs in breast cancer cells was analyzed using the DCFH-DA assay (Fig. 7A). DCFH, a non-fluorescent dye, reacts with singlet oxygen and generate free radicals upon treatment with MNPs, converting DCFH into the green fluorescent compound DCF, which indicates oxidative stress and apoptosis.⁸⁵ In our study, both BMNP and CMNP treatments significantly increased ROS levels, as evidenced by enhanced green fluorescence in breast cancer cells (MCF-7 and MDA-MB-231). Notably, CMNPs exhibited improved cellular localization and uptake, which amplified oxidative stress, leading to apoptosis via mitochondrial dysfunction, which requires further investigation. To understand the underlying mechanism of apoptosis via mitochondrial dysfunction, Rhodamine 123 staining was performed (Fig. 7B). Rhodamine 123, a negatively charged dye, preferentially binds to healthy, polarized mitochondria, emitting green fluorescence.⁸⁶ The results from the study revealed that the control cells exhibited increased green fluorescence compared to the cells treated with BMNPs and CMNPs. The reduced fluorescence in the treated cells indicates a loss of mitochondrial membrane potential, signifying mitochondrial dysfunction and apoptosis induction mediated by mitochondrial pathways.⁴³

Fig. 7. BMNPs and CMNPs induce oxidative stress (A) and mitochondrial dysfunction (B) in breast cancer cells. (A) Intracellular reactive oxygen species (ROS) generation assessed by DCFH-DA staining in MCF-7 and MDA-MB-231 cells treated with BMNPs (ii and v) and CMNPs (iii and vi) at their IC₅₀ concentrations for 24 h. (i and iv) Untreated cells show minimal fluorescence, while the BMNP- and CMNP-treated cells exhibit markedly increased ROS levels. (vii) Corresponding fluorescence quantification. (B) Mitochondrial membrane potential (MMP) analysis in MCF-7 and MDA-MB-231 cells following BMNP (ii and v) and CMNP (iii and vi) treatments. (i and iv) Untreated cells display intact mitochondrial integrity, while the treated cells show a loss of MMP, indicated by altered mitochondrial fluorescence. (vii) Quantitative analysis of MMP disruption. Data are expressed as mean ± SD (n = 3).

Further, BMNPs and CMNPs were evaluated for their effects on the migratory behavior of MCF-7 and MDA-MB-231 breast cancer cells using wound-healing (scratch) assays.⁸⁷ In untreated controls, both cell lines exhibited regular wound closure over 0–48 hours, confirming their intrinsic migratory capabilities. In our study, BMNP-treated MCF-7 and MDA-MB-231 cells displayed restrained migration (SI Fig. 11). Visual inspection of the scratch assays showed prominent gaps persisting up to 48 hours in the treated samples, while the controls demonstrated rapid wound closure. For CMNPs, both MCF-7 and MDA-MB-231 cells showed significantly slower wound closure rates than those observed for BMNPs, which indicate the existence of prominent gaps in the treated groups up to 48 h, compared to near-complete wound closure in the controls (Fig. 8). The strong suppression in both cell lines suggests that CMNPs interfere intensively with cytoskeletal reorganization or signaling pathways critical for cell motility.^88,89 Hence, it is clear from the study that CMNPs are highly effective in impeding breast cancer migration, with nearly complete suppression in both luminal (MCF-7) and triple-negative (MDA-MB-231) sub-types.

Fig. 8. Scratch wound healing assay showing the effect of CMNPs on the migration of MCF-7 and MDA-MB-231 breast cancer cells. Representative phase-contrast images were acquired at 0, 12, 24, 36, and 48 h after scratch formation. (a–e) Untreated MCF-7 control cells; (f–j) CMNP-treated MCF-7 cells; (k–o) untreated MDA-MB-231 control cells; and (p–t) CMNP-treated MDA-MB-231 cells. Quantitative analysis of wound closure for (u) MCF-7 cells and (v) MDA-MB-231 cells. Cell migration is expressed as the percentage of wound closure relative to the initial wound width (0 h). Data are presented as mean ± SD (or SE, as applicable) from three independent experiments. Statistical significance was determined using one-way ANOVA followed by an appropriate post hoc test. p < 0.05, *p < 0.01, **p < 0.001, ***p < 0.0001; ns, not significant.

In the present study, Prussian blue staining was employed to detect ferric iron and confirm the intracellular uptake of both BMNPs (50 µg mL⁻¹) and CMNPs (50 µg mL⁻¹) in both MCF-7 and MDA-MB-231 cells, respectively.⁹⁰ In the untreated (control) groups, both MCF-7 (a) and MDA-MB-231 (e) cells exhibited minimal or no visible blue coloration, indicating the absence of intracellular iron deposits (SI Fig. 12). Following exposure to BMNPs and CMNPs, a marked increase in dark blue staining within the cytoplasm was observed in both MCF-7 (SI Fig. 12b) and MDA-MB-231 (SI Fig. 12f) cells, confirming the internalization of iron-containing nanoparticles. In our study, CMNPs showed more intense dark blue coloration than BMNPs, highlighting greater cellular uptake, which may be attributed to the colloidal stability aided by citrate capping. A similar observation has been reported for citrate-coated iron oxide nanoparticles, where enhanced dispersion facilitates efficient cellular internalization.^91,92

Flow cytometry analysis demonstrates that both BMNPs and CMNPs show notable alterations in the cell cycle profiles of MCF-7 and MDA-MB-231 breast cancer cells compared to the control groups, as shown in Fig. 9.⁹³ In both MCF-7 and MDA-MB-231 cells, the control (untreated) cells display the typical cell-cycle distribution, with the majority of cells in the G1 phase, moderate populations in the S phase, and a minor percentage of G2 phase. Upon treatment with BMNPs, a significant increase in the G1 cell population (MCF-7: 60.9% and MDA-MB-231: 62%) was observed relative to the controls, accompanied by modest reductions in S (MCF-7: 16.2% and MDA-MB-231: 13.9%) and G2 (MCF-7: 3.52% and MDA-MB-231: 3.74%) populations. Meanwhile, CMNPs result in a dramatic elevation of G1-phase cells, particularly in MCF-7 (97%), and a clearly enhanced G1-phase in MDA-MB-231 (82.9%), combined with a sharp depletion of both S (MCF-7: 4.79% and MDA-MB-231: 15%) and G2 (MCF-7: 1.58% and MDA-MB-231: 5.86%) phases. Bar graphs further highlight these shifts, showing G1 accumulation as the dominant response, especially for CMNPs.

Fig. 9. BMNPs and CMNPs induce cell cycle arrest and apoptosis in breast cancer cells. Flow cytometric cell cycle analysis of propidium iodide–stained MCF-7 (a–d) and MDA-MB-231 (e–h) cells following 24 h of treatment. Untreated MCF-7 (a) and MDA-MB-231 (e) cells, respectively; MCF-7 (b) and MDA-MB-231 (f) cells treated with BMNPs; and MCF-7 (c) and MDA-MB-231 (g) cells treated with CMNPs. Histograms show the distribution of cells across the G0/G1, S, and G2/M phases, with the sub-G0/G1 population representing MCF-7 (d) and MDA-MB-231 (h) apoptotic cells.

These quantitative trends correlate with the previously documented attributes of surface-modified magnetic nanoparticles concerning the promotion of cell cycle arrest in the G1 or S phases, particularly in the inhibition of DNA synthesis in cancer cell systems.⁹⁴ The induction of G1 arrest by BMNPs and CMNPs corresponds to the interference of the regulatory checkpoint proteins and cyclin-dependent kinases that control the G1 to S phase progression.³⁴ This influence seems to be more prominent with citrate stabilization and may stem from its high colloidal stability, supposed increase in cellular uptake, and the surface charge interactions in receptor and intracellular targets.^95,96 These observations reinforce the ability of CMNPs to induce G1 cell cycle arrest and suggest that surface functionalization significantly influences the intracellular response in breast cancer cells.

In order to study the induction of apoptosis in MCF-7 and MDA-MB-231 cells by BMNPs and CMNPs, Annexin V/PI flow cytometric analysis was carried out (Fig. 10). Annexin V/PI flow cytometric analysis demonstrated that both BMNPs and CMNPs induced substantial apoptosis in MCF-7 and MDA-MB-231 cells. In our study, BMNP treatment increased the early and late apoptotic populations to 39.35% and 12.70%, respectively, while CMNPs yielded 20.62% early apoptosis and 30.78% late apoptosis in MCF-7 cells. Similarly, in MDA-MB-231 cells, BMNP treatment reduced the viable population to 45.50%, with 44.38% early apoptosis and 5.05% late apoptosis, whereas CMNPs produced 43.97% early apoptosis and 14.08% late apoptosis. A marked reduction in viable cells and the accumulation of Annexin V-positive populations confirm that both BMNPs and CMNPS primarily trigger apoptotic cell death.⁹⁷

Fig. 10. Annexin V-FITC/PI flow cytometric analysis of apoptosis in MCF-7 and MDA-MB-231 cells following BMNP and CMNP treatments. Representative dot plots of (a–c) MCF-7 cells (control, BMNPs, and CMNPs, respectively) and (d–f) MDA-MB-231 cells (control, BMNPs, and CMNPs, respectively). Quadrants represent viable (B3), early apoptotic (B4), late apoptotic (B2), and necrotic (B1) cell populations.

It is also noted from the study that CMNPs induced a greater proportion of apoptotic cell death in MCF-7 cells, suggesting that citrate surface functionalization imparts apoptotic potency. Meanwhile, a slight increase in the necrotic cell population in MDA-MB-231 was noticed during the treatment with BMNPs and CMNPs. However, this minimal increase is likely attributable to secondary necrosis following apoptosis rather than primary necrotic cell death. In light of this, MDA-MB-231 cells exhibited predominant early and late apoptotic cell populations than the necrotic cells authenticating apoptosis as the principal mechanism underlying BMNP- and CMNP-induced cytotoxicity. Based on the observation, the results obtained from the study are consistent with an ROS-driven mitochondrial pathway of apoptosis, which caused mitochondrial depolarization and ROS generation, as revealed by fluorescence microscopic analysis. Moreover, the above-mentioned alterations collectively support the activation of the intrinsic mitochondrial apoptotic pathway targeting MCF-7 and MDA-MB-231 breast cancer cells, which requires further investigation.

Based on the preliminary findings, both BMNPs and CMNPs induced cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells by inhibiting cell proliferation, restricting cell migration, and causing cell cycle arrest. However, the exact molecular mechanism by which BMNPs and CMNPs inhibit breast cancer cells is not yet known. Due to this, the impact of BMNPs and CMNPs on the induction of apoptosis in breast cancer cell lines (MCF-7 and MDA-MB-231) was carefully analyzed using qRT-PCR and western blot studies (Fig. 11 and 12, respectively). The expression patterns of crucial apoptotic genes and proteins were evaluated, providing insights into the mechanistic pathways involved in the therapeutic efficacy of these nanoparticles (SI Fig. 9).

Fig. 11. qRT-PCR analysis of apoptosis- and survival-related genes in breast cancer cells following BMNP and CMNP treatments. Relative mRNA expression of AKT, PI3K, PTEN, Bcl-2, cytochrome c (Cyto C), and caspase-9 in (a) MCF-7 and (b) MDA-MB-231 cells after treatment with BMNPs and CMNPs. Gene expression was normalized to GAPDH and is presented as fold change relative to the untreated control. Data are expressed as mean ± SD (or SE, as applicable) from three independent experiments. Statistical significance: p < 0.05, *p < 0.01, **p < 0.001, ***p < 0.0001; ns, not significant.

Fig. 12. Western blot analysis of apoptosis- and survival-related proteins in breast cancer cells following BMNP and CMNP treatments. Representative immunoblots and corresponding densitometric analysis of p-AKT, PI3K, cytochrome c (Cyto C), and caspase-9 in (a) MCF-7 and (c) MDA-MB-231 cells after treatment with BMNPs and CMNPs. GAPDH was used as the loading control. Protein expression is presented as fold change relative to the untreated control for MCF-7 (b) and MDA-MB-231 (d). Data are expressed as mean ± SD (or SE, as applicable) from three independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

It is a well-known fact that cancer cells exhibit rapid cell proliferation by altering a few key signaling elements, which are crucial factors in regulating normal cellular processes.⁹⁸ In view of this, PI3K/AKT signaling remains a key player in regulating a cascade of signaling involving AKT, also known as protein kinase B, as a central mediator of PI3K (phosphatidylinositol 3-kinase), while PTEN (phosphatase and tensin homolog) functions as a tumor suppressor and a negative regulator of this pathway.^99,100 As shown in Fig. 11, the gene expression patterns of PI3K and AKT were evaluated following treatment with BMNPs and CMNPs in MCF-7 and MDA-MB-231 breast cancer cells via RT-qPCR. Our results demonstrate that the MCF-7 and MDA-MB-231 breast cancer cells showed a significant downregulation of PI3K and AKT mRNA expression levels compared with untreated controls. At the protein level, western blot analysis corroborates the findings of the gene expression studies by demonstrating a visible downregulation of PI3K and p-AKT signals in both breast cancer cell lines following BMNP and CMNP treatment, confirming the prevention of cell proliferation at the gene and protein levels (Fig. 12).

The suppression of PI3K/AKT signaling by MNPs suggests a disruption of pro-survival signals, thereby sensitizing cancer cells to apoptotic stimuli.¹⁰¹ Notably, the downregulation of PI3K/AKT genes and proteins was more pronounced with CMNPs, indicating their superior efficacy in targeting this oncogenic pathway. In parallel, the tumor suppressor gene PTEN, a critical negative regulator of the PI3K/AKT signaling pathway, exhibited significant upregulation at the mRNA level following treatment with BMNPs and CMNPs (Fig. 11). The increased expression of PTEN further supports the inhibition of PI3K/AKT signaling and reinforces the pro-apoptotic effects of the MNPs.¹⁰² In our study, the greater fold increase in PTEN expression observed with CMNP-treated cells suggests that citrate stabilization enhances the therapeutic efficacy of MNPs, highlighting a potentially novel mechanistic advantage, which has not been previously reported.

Furthermore, the effect of both BMNPs and CMNPs on mitochondrial membrane potential (ΔΨ_m) was investigated on MCF-7 and MDA-MB-231 breast cancer cells. In our study, the anti-apoptotic gene Bcl-2 showed a relative decrease in gene expression pattern following treatment with BMNPs and CMNPs (Fig. 11).¹⁰³ In the literature, Bcl-2 is a key regulator of mitochondrial integrity, and its downregulation is known to facilitate mitochondrial outer membrane permeabilization (MOMP).¹⁰⁴ Subsequent to MOMP, cytochrome c is released from mitochondria into the cytosol, where it promotes apoptosome formation and subsequent activation of caspase-9, culminating in apoptosis.¹⁰⁵ In agreement with this mechanism, we observed significant upregulation of cytochrome c and caspase-9 at the gene and protein levels, demonstrating the activation of the intrinsic mitochondrial apoptotic pathway (Fig. 11 and 12).^106–108 Notably, CMNPs exhibited a greater degree of pro-apoptotic effects than BMNPs, suggesting that CMNPs are more potent activators of mitochondrial-mediated apoptosis.

In our study, CMNPs consistently demonstrated superior efficacy across all analyzed pro-apoptotic genes compared to BMNPs. The enhanced activity of CMNPs can be attributed to the surface modification conferred by citrate, which may amplify the apoptotic signaling pathways. The observed outcomes align with preliminary evidence, including nuclear condensation, increased reactive oxygen species (ROS) generation, mitochondrial membrane damage, reduced cell migration, and cell cycle arrest. These findings suggest that BMNPs and CMNPs effectively induce apoptosis in breast cancer cells by modulating the PI3K/AKT/PTEN pathway and thereby triggering the apoptotic cascade in in vitro pre-clinical models. The superior pro-apoptotic activity exhibited by CMNPs highlights their potential as nanotherapeutics for targeted breast cancer treatment. However, in vivo studies are required to validate the translational potential of CMNPs for clinical applications.

Author contributions

S. D. performed all experiments under the mentorship of P. K. P. K. wrote and critically reviewed the manuscript. P. K. also acquired funding to execute this project.

Conflicts of interest

The authors declare no competing interests.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03590f.