Introduction
Cancer is one of the primary causes of death globally, with conventional treatment strategies often compromised by lack of specificity, multidrug resistance, and severe side effects1. Developing innovative therapeutic approaches that selectively target cancer cells while sparing normal tissues represents a critical frontier in cancer research2. Nanotechnology offers potential for developing novel anticancer strategies, with magnetic nanoparticles (NPs) drawing particular attention due to their unique physicochemical properties and multifunctional capabilities3, 4–5.
The hematological cancer known as multiple myeloma (MM) is characterized by the aberrant proliferation of bone marrow plasma cells, resulting in monoclonal immunoglobulins and consequent organ damage6. Despite recent advances in treatment modalities, including proteasome inhibitors, immunomodulatory drugs, and stem cell transplantation, multiple myeloma remains incurable primarily, with most patients experiencing relapse and developing drug resistance7. The U266 cell line represents a well-established in vitro model for studying multiple myeloma, exhibiting characteristic features such as elevated interleukin-6 (IL-6) and immunoglobulins8. Acute monocytic leukemia (AML-M5) is an aggressive subtype of acute myeloid leukemia characterized by a rapid accumulation of abnormal monocytes in the bone marrow and peripheral blood, leading to impaired hematopoiesis and organ infiltration9. Treatment typically involves intensive chemotherapy regimens, which are often associated with significant toxicity and variable outcomes, particularly in older patients or those with high-risk genetic profiles9. The THP-1 cell line is a valuable model for investigating acute monocytic leukemia, offering insights into leukemogenesis, immune function, and potential therapeutic targets10,11. Both multiple myeloma and acute monocytic leukemia present significant therapeutic challenges due to their complex pathophysiology, heterogeneity, and propensity for developing treatment resistance. Novel therapeutic approaches that selectively target these malignancies while minimizing toxicity to normal cells are critically required to enhance patient outcomes12.
Magnetite (Fe₃O₄) NPs have garnered significant interest in biomedical applications owing to their superparamagnetic properties, biocompatibility, and tunable surface chemistry13. These NPs offer therapeutic advantages, including targeted drug delivery, magnetic hyperthermia, and magnetic resonance imaging capabilities14. However, conventional synthesis methods for magnetite NPs typically involve toxic chemicals, harsh reaction conditions, and environmentally harmful processes, which raise concerns regarding their biocompatibility and ecological impact15. Solvo-/hydrothermal synthesis methodologies offer a cost-effective and versatile method for manufacturing large quantities of highly crystalline magnetic clusters with controlled morphology16, 17–18. They can modulate both primary and secondary cluster dimensions, determining the magnetic characteristics of the particles. Green synthesis approaches using plant extracts have become sustainable alternatives for nanoparticle production, eliminating the need for hazardous chemicals while potentially enhancing biocompatibility through natural surface functionalization19, 20–21. Recent advances in magnetic NP synthesis highlight the growing interest in green methods for biomedical applications, providing a foundation for the microwave-solvothermal approach.
Among the various plant sources, Hippophae rhamnoides (sea buckthorn) berries present a candidate for green synthesis due to their remarkable phytochemical profile, including high concentrations of vitamin C, flavonoids, phenolic compounds, and organic acids with potent antioxidant and potential anticancer properties22,23. Sea buckthorn (H. rhamnoides) has been traditionally utilized in various medical systems, particularly in Tibetan, Mongolian, and Chinese medicine, for treating various ailments, including cancer, cardiovascular diseases, and inflammatory conditions24,25. The integration of sea buckthorn’s therapeutic properties with the intrinsic capabilities of magnetite NPs presents an innovative approach to developing natural nano-chemotherapeutic agents. Despite the growing interest in green-synthesized magnetic NPs, comprehensive studies evaluating their anticancer mechanisms, particularly their selective cytotoxicity against different cancer cell types compared to normal cells, remain limited. Understanding these aspects is crucial for advancing their potential clinical applications while ensuring safety and efficacy.
Previous studies have reported green synthesis of Fe₃O₄-based NPs using plant extracts like Eryngium planum L26. and Mentha longifolia27, focusing on their applications in medicine and cancer therapy. Our study advances this field by using H. rhamnoides berry extract in a microwave-solvothermal synthesis, combining rapid synthesis with natural surface functionalization to enhance biocompatibility and selective anticancer efficacy against multiple myeloma (U266) and acute monocytic leukemia (THP-1) cell lines compared to normal fibroblasts (L-929). The research investigates the physicochemical properties of the synthesized NPs through multiple characterization techniques and elucidates their anticancer mechanisms through comprehensive in vitro assays, including cell viability, proliferation, apoptosis, and oxidative stress assessment. The findings provide valuable insights into the potential of these green-synthesized NPs as selective anticancer agents and their underlying mechanisms of action.
Materials and methods
Materials
Hippophae rhamnoides (sea buckthorn) berries were commercially sourced from local markets. Iron (III) chloride hexahydrate (FeCl₃·6 H₂O) and iron (II) chloride tetrahydrate (FeCl₂·4 H₂O) were acquired from Merck Company and utilized without further purification. Cell culture reagents, including penicillin-streptomycin, fetal bovine serum (FBS), RPMI 1640, and Dulbecco’s Modified Eagle Medium (DMEM), were purchased from Sigma Aldrich. Human Caspase 3 (Cleaved) ELISA Kit was procured from Invitrogen, while antioxidant assay kits were obtained from Navand Salamat, Iran. The Iranian Arshanzist Youtab Company (Kerman, Iran) generously provided all additional materials.
Hippophae rhamnoides (sea buckthorn) berries extract Preparation
Fresh H. rhamnoides berries were washed thoroughly with deionized water, dried, and ground to a fine paste. The paste was mixed with deionized water (1:5 w/v ratio) and heated at 80 °C for 30 min under constant stirring. The mixture was filtered through Whatman No. 1 filter paper, and the obtained extract was stored at 4 °C until further use. The phytochemicals in the sea buckthorn extract, particularly polyphenols, flavonoids, and ascorbic acid, are well-documented in the literature22,28,29 and are known to serve as reducing and capping agents in green synthesis30,31.
Synthesis and characterization of fe₃o₄ NPs
Fe₃O₄ NPs were synthesized using a green microwave-assisted method with Hippophae rhamnoides (sea buckthorn) berries extract. For NPs synthesis, 2.70 g of iron (III) chloride hexahydrate (FeCl₃·6 H₂O) and 0.99 g of iron (II) chloride tetrahydrate (FeCl₂·4 H₂O) (molar ratio 2:1) were dissolved in 50 mL of distilled water. Separately, 10 mL of H. rhamnoides extract was diluted with 40 mL of distilled water and added dropwise to the iron salt solution under stirring at 500 rpm for 30 min. The pH was adjusted to 10 using 1 M NaOH, forming a brownish-black solution. This solution was irradiated at 900 W for 5 min in a microwave reactor (2.45 GHz), then transferred to a 100 mL Teflon-lined autoclave and heated at 150 °C for 24 h. These parameters were optimized through preliminary experiments: microwave power below 900 W yielded incomplete reduction, while higher power caused aggregation; solvothermal durations shorter than 24 h resulted in mixed iron oxide phases. The product was washed, centrifuged at 5000 rpm for 10 min, magnetically separated, and vacuum-dried at 80 °C for 24 h. Reproducibility was confirmed across two batches, yielding consistent primary sizes, and magnetization. The biosynthesized Fe₃O₄ NPs were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), Transmission Electron Microscope (TEM), Fourier-transform infrared spectroscopy (FTIR), and vibrating sample magnetometry (VSM) to confirm their crystalline structure, morphology, size distribution, surface functionalization by plant metabolites, and magnetic properties, respectively.
Cell culture
Human multiple myeloma (U266), acute monocytic leukemia (THP-1), and mouse fibroblast (L-929) cell lines were obtained from the Pasteur Institute collection in Tehran, Iran. The Pasteur Institute maintains rigorous cell line authentication protocols, with comprehensive catalogs accessible via their official repository (https://en.pasteur.ac.ir/Department-of-Cell-Bank). Our laboratory has established a consistent procurement relationship with the Pasteur Institute, resulting in numerous peer-reviewed publications utilizing these authenticated cell lines32, 33, 34–35. U266 and THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. L-929 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with the same supplements. All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were periodically tested for mycoplasma contamination using the MycoAlert® Mycoplasma Detection Kit.
Cell viability assay
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and allowed to attach for 24 h. Cells were then treated with various concentrations (25, 50, 75, 100, and 150 µg/mL) of Fe₃O₄ NPs for 24 and 48 h. The concentration range of 25–150 µg/mL was selected based on previous studies and pilot experiments20,36, which showed minimal cytotoxicity below 25 µg/mL and non-specific toxicity above 150 µg/mL, allowing for dose-dependent analysis while maintaining selectivity. Control cells received an equivalent volume of the vehicle. After treatment, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well, and plates were incubated for another 4 h at 37 °C. The formazan crystals formed were solubilized by adding 100 µL of dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage relative to the untreated control cells37.
Cell proliferation assay
Cell proliferation was evaluated using the 5-bromo-2’-deoxyuridine (BrdU) incorporation assay according to the manufacturer’s instructions. Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and treated with Fe₃O₄ NPs at concentrations ranging from 25 to 150 µg/mL for 24 and 48 h. BrdU was added to the culture medium 4 h before the end of the treatment period. After fixation and DNA denaturation, cells were incubated with anti-BrdU antibody conjugated with peroxidase. The immune complexes were identified by measuring absorbance at 450 nm using a microplate reader. The findings were presented as optical density (OD) values, representing the quantity of BrdU integrated into the freshly manufactured DNA.
Apoptosis detection
Apoptosis was assessed using the Annexin V-FITC/propidium iodide (PI) double staining method. Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and treated with 75 and 150 µg/mL of Fe₃O₄ NPs for 48 h. After treatment, cells were harvested, washed, and stained with Annexin V-FITC and PI using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s protocol. Data were analyzed using FlowJo software. Cells were classified as viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), or necrotic (Annexin V⁻/PI⁺). The percentage of cells in each category was calculated. Each sample was assayed in triplicate, and the experiment was repeated 3 times.
Caspase-3 activity assay
The activity of caspase-3 was measured using the Caspase-3 Colorimetric Assay Kit (Invitrogen). Cells were treated with Fe₃O₄ NPs at concentrations ranging from 25 to 100 µg/mL for 48 h. Following treatment, cells were harvested and lysed in the lysis buffer provided in the kit. The activity of caspase-3 was determined for each well according to the manufacturer’s protocol. Each sample was assayed in triplicate, and the experiment was repeated 3 times.
Oxidative stress assessment
Total oxidant status (TOS) was determined using a colorimetric assay based on the oxidation of ferrous ions to ferric ions in the presence of various oxidant species. Briefly, cells were treated with Fe₃O₄ NPs (25–100 µg/mL) for 48 h, harvested, and lysed. Cell lysates were mixed with reagents 1 (xylenol orange, NaCl, and glycerol) and 2 (ferrous ion and o-dianisidine in H₂SO₄ solution). After incubation, the absorbance was measured at 560 nm. Results were expressed as µmol H₂O₂ equivalent per liter (µmol H₂O₂ Equiv./L)38. Each sample was assayed in triplicate, and the experiment was repeated 3 times.
Lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels. Treated cells were harvested and homogenized. The homogenate was mixed with 0.67% thiobarbituric acid (TBA) and 20% trichloroacetic acid solution and heated at 100 °C for 30 min. After cooling, the mixture was centrifuged, and the absorbance of the supernatant was measured at 532 nm. MDA concentration was calculated using the molar extinction coefficient of the MDA-TBA complex (1.56 × 10⁵ M⁻¹cm⁻¹) and expressed as nmol/mg protein38. Each sample was assayed in triplicate, and the experiment was repeated 3 times.
Statistical analysis
All experiments were performed in triplicate and repeated at least three times. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software. Differences between the control and treatment groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Statistical significance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. IC₅₀ values (half-maximal inhibitory concentration) were calculated using nonlinear regression analysis.
Results and discussion
X-ray diffraction analysis
Figure 1 presents the X-ray diffraction (XRD) pattern of the synthesized Fe₃O₄ NPs. The X-ray Diffraction (XRD) pattern of the green-synthesized Fe₃O₄ NPs, recorded using Cu Kα radiation, provides insights into the crystalline structure and phase composition, with diffraction peaks plotted as counts against the 2θ angle ranging from 10° to 80°. Prominent diffraction peaks are observed at 2θ values of 31.1°, 40.0°, 42.2°, 51.1°, and 44.0°, which are consistent with the characteristic reflections of the Fe₃O₄ (magnetite) phase as per JCPDS Card No. 88–031539. According to this card, the standard peak positions for Fe₃O₄ include 30.1° for the (220) plane, 35.4° for the (311) plane, 43.1° for the (400) plane, 53.4° for the (422) plane, and 56.9° for the (511) plane. The observed peak at 31.1° aligns closely with the (220) plane, the peak at 40.0° corresponds to the (311) plane with a slight shift, and the peak at 51.1° is consistent with the (400) plane. The intensity of the (311) peak at 40.0°, with the highest count of approximately 400, is the most prominent, which is typical for magnetite NPs and aligns with the relative intensity trends in JCPDS 88–0315. The slight deviations in 2θ positions (e.g., 40.0° vs. 35.4° for (311)) may be attributed to lattice strain, crystallite size effects, or the influence of the organic capping agents from the Hippophae rhamnoides berry extract40, 41, 42–43. The sharpness of the peaks suggests good crystallinity, while their broad nature, particularly at 31.1° and 40.0°, indicates nanoscale crystallite sizes, typical for green-synthesized NPs. The crystallite size can be estimated using the Scherrer Eq. (42). For the (311) peak at 40.0° (2θ), an approximate FWHM of 0.5° (converted to radians as 0.0087), the crystallite size is approximately , confirming that the green synthesis method yields Fe₃O₄ NPs in the nanoscale range. The absence of additional peaks related to other iron oxide phases or significant organic residues suggests phase purity, with the organic components likely acting as amorphous capping agents below the detection limit of XRD.
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Fig. 1
XRD patterns of green synthesized Fe₃O₄ NPs with Hippophae rhamnoides berry extract.
Fourier transform infrared analysis
Figure 2 shows the Fourier Transform Infrared (FTIR) spectrum of the Fe₃O₄ NPs. The FTIR spectra of Fe₃O₄ NPs synthesized via the conventional NaOH method (a) and the green synthesis method using Hippophae rhamnoides berry extract (b) were analyzed in the wavenumber range of 3500 to 500 cm⁻¹ to elucidate their surface chemistry and functional group composition. In spectrum (a), representing the NaOH-synthesized Fe₃O₄ NPs, a broad O-H Stretching Band at 3440.94 cm⁻¹ is observed, indicative of O-H stretching vibrations from residual water molecules or surface hydroxyl groups, with its broadness suggesting hydrogen bonding44. A weaker O-H Bending Band at 1636.90 cm⁻¹ further confirms the presence of adsorbed water or hydroxyl groups on the NP surface45,46. The absence of significant absorption in the 3000–2500 cm⁻¹ region highlights the lack of organic C-H groups, consistent with the inorganic nature of the NaOH synthesis method47,48. A band at 1089.73 cm⁻¹, identified as the Fe-O stretching vibrations, which serve as a clear indicator of iron oxide49. Another band at 809.04 cm⁻¹, designated as the Fe-O-H Bending Band, is likely associated with bending vibrations of Fe-O-H groups on the nanoparticle surface, reflecting possible hydroxylation of the Fe₃O₄ surface during synthesis in an aqueous NaOH environment50,51. A Fe-O Stretching Band at 585.53 cm⁻¹ is a hallmark of the Fe₃O₄ structure, reflecting its well-crystallized magnetite core52,53. A secondary Fe-O band appears at 466.03 cm⁻¹, which is typically associated with octahedral sites in the Fe₃O₄ structure 54.
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Fig. 2
FTIR spectra comparing Fe₃O₄ NPs synthesized using NaOH (a) and the green method with Hippophae rhamnoides berry extract (b).
In contrast, spectrum (b) of the green-synthesized Fe₃O₄ NPs reveals additional organic signatures due to the Hippophae rhamnoides berry extract. Although the FTIR spectrum of the extract itself is unavailable in our study, literature reports22,28,29 indicate that H. rhamnoides berries contain polyphenols, flavonoids, and ascorbic acid, which typically exhibit these functional groups and are known to act as reducing and capping agents in green synthesis55, 56–57. The broad absorption bands observed at 3572.41, 3488.35, and 3388.48 cm⁻¹ correspond to O-H stretching vibrations, attributed to surface-adsorbed water molecules, hydroxyl groups on the magnetite surface, and phenolic/alcoholic hydroxyl groups derived from bioactive compounds in the Hippophae rhamnoides berry extract that remain bound to the nanoparticle surface during the green synthesis process43,58. The absorption bands in the mid-infrared region at 1152.42, 1111.06, 1082.63, and 987.89 cm⁻¹ correspond to C-O stretching vibrations and skeletal vibrations of organic molecules from the berry extract, indicating successful incorporation of bioactive compounds such as flavonoids, phenolic acids, and polysaccharides onto the Fe₃O₄ surface, which act as natural capping and stabilizing agents55, 56–57,59. The peaks at 888.03, 853.58, 802.04, and 757.73 cm⁻¹ represent C-O-C stretching and bending vibrations of glycosidic bonds and aromatic C-H bending modes from the organic matrix derived from the plant extract60, 61–62. The characteristic absorption bands at 642.08, 608.67, 519.67, 489.92, and 423.27 cm⁻¹ represent the Fe-O stretching and Fe-O-Fe bending vibrations typical of the inverse spinel structure of magnetite (Fe₃O₄)63,64, with the bands around 642.08 and 519.67 cm⁻¹ being particularly diagnostic for tetrahedral and octahedral Fe-O bonds in the magnetite lattice, confirming the successful formation of crystalline iron oxide NPs50,65,66. The presence of multiple absorption bands throughout the spectrum demonstrates that the Hippophae rhamnoides berry extract components effectively function as both reducing agents for iron salt reduction and capping agents for nanoparticle stabilization, creating a biocompatible organic corona around the magnetic core that enhances the bioactivity, stability, and potential therapeutic applications of the synthesized Fe₃O₄ NPs. The slight shift and broadening of the Fe-O bands compared to spectrum (a) may result from the organic coating influencing the magnetite lattice. Comparatively, the NaOH method produces Fe₃O₄ NPs with a predominantly inorganic character, albeit with minor impurities or surface modifications as indicated by the S-O and Fe-O-H bands. In contrast, the green synthesis method introduces a richer array of organic functional groups, enhancing the NPs’ surface functionality, which could improve their biocompatibility or dispersibility for specific applications67,68.
Mechanism of fe₃o₄ nanoparticle formation
The formation of Fe₃O₄ NPs likely proceeds through the following steps1: Complexation of Fe³⁺ and Fe²⁺ ions with phytochemicals (e.g., polyphenols, flavonoids) in the berry extract, acting as chelating agents2; Reduction of Fe³⁺ to Fe²⁺ by the antioxidant properties of ascorbic acid and polyphenols, facilitating co-precipitation at pH 103; Nucleation and growth accelerated by microwave irradiation, with phytochemicals controlling particle size; and4 Surface functionalization by adsorption of biomolecules, enhancing biocompatibility. This mechanism is inferred from the FTIR data of the green-synthesized NPs (Fig. 2b), which show organic functional groups and is consistent with green synthesis mechanisms reported in the literature69,70. The H. rhamnoides berry extract contains polyphenols, flavonoids, and ascorbic acid, which play distinct roles in the synthesis22,28,29. Polyphenols reduce Fe³⁺ to Fe²⁺ through electron donation from their hydroxyl groups while stabilizing the NPs by adsorbing onto their surface, as inferred from the FTIR spectrum (Fig. 2b)30,31. These roles are supported by organic functional groups on the NP surface, confirming the extract’s contribution to the synthesis process. Figure 3 presents a schematic of the Fe₃O₄ NP formation mechanism, illustrating the steps of complexation, reduction, nucleation, and surface functionalization.
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Fig. 3
Mechanism of Fe₃O₄ NPs Formation.
Energy dispersive X-ray analysis
Figure 4 presents the Energy Dispersive X-ray (EDX) spectroscopy analysis of the synthesized Fe₃O₄ NPs using Hippophae rhamnoides berry extract. The spectrum displays characteristic elemental peaks that provide valuable information about the chemical composition of the NPs71. Strong peaks corresponding to iron (Fe) are visible, with the Fe Kα peak at approximately 6.4 keV and the Fe Kβ peak at around 7.1 keV, confirming the presence of iron as the principal metallic element in the NPs. The spectrum also exhibits a prominent oxygen (O) Kα peak at approximately 0.53 keV, which validates the formation of iron oxide. Additionally, a carbon (C) Kα peak is observed at about 0.28 keV, attributable to the organic compounds from the Hippophae rhamnoides berry extract that function as capping and stabilizing agents on the nanoparticle surface. The relative intensities of these peaks suggest that the NPs possess a stoichiometry close to the theoretical Fe: O ratio of 3:4 in magnetite (Fe₃O₄). However, precise quantification would require the integration of peak areas. The absence of significant impurity peaks indicates the high purity of the synthesized NPs, which is consistent with the XRD results. The presence of carbon further substantiates the FTIR findings, confirming that bioactive compounds from the berry extract have been successfully incorporated into the NP’s surface. Overall, the EDX analysis confirms the elemental composition of the green-synthesized Fe₃O₄ NPs and supports their successful preparation using the Hippophae rhamnoides berry extract as an eco-friendly reducing and capping agent72.
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Fig. 4
The EDX spectroscopy analysis of the synthesized Fe₃O₄ NPs confirms the presence of Fe and O.
Scanning Electron microscopy morphological studies
Figure 5 presents SEM and TEM micrographs of the green-synthesized Fe₃O₄ NPs. Figures (a-c) show SEM views at 1 μm, 500 nm, and 500 nm scales, respectively, with high-quality scale markers for accurate size assessment. Figure 5a, captured at a lower magnification (scale bar: 1 μm), reveals an overall agglomerated structure consisting of numerous small, quasi-spherical particles clustered into larger secondary structures. These agglomerates display a rough surface texture formed by the assembly of primary NPs. Figure 5b shows a higher magnification view of the nanoparticle agglomerates, demonstrating their irregular three-dimensional architecture with visible interparticle voids and crevices, which could potentially enhance the surface area available for biological interactions. Figure 5c focuses on specific agglomerated clusters, clearly showing their composite nature formed by the fusion of smaller primary particles. The distinct boundaries between aggregated structures are visible, suggesting that the individual particles maintain some degree of autonomy within the clusters. The surface of the agglomerates appears granular, with the primary NPs exhibiting semi-spherical to polyhedral shapes. This hierarchical organization of NPs into larger structures is likely influenced by the phytochemicals from the Hippophae rhamnoides berry extract that act as natural capping agents23,43. These structures’ nanoscale roughness and high surface area may contribute to their enhanced biological activity, particularly their selective cytotoxicity against cancer cells73,74. Overall, the SEM analysis confirms the successful synthesis of Fe₃O₄ NPs with complex hierarchical morphology, where primary NPs assemble into larger secondary structures, a characteristic often observed in green-synthesized nanomaterials due to the influence of biomolecules in the plant extract58,75. Figure 5d is a TEM micrograph at a 60 nm scale, providing a high-resolution view of the primary NPs. The TEM image reveals primary NPs with sizes ranging from 15.6 nm, confirming their nanoscale dimensions.
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Fig. 5
SEM and TEM images of Fe₃O₄ NPs at different magnifications. (a) SEM at 1 μm scale, (b) SEM at 500 nm scale, (c) SEM at 500 nm scale, showing agglomerates, (d) TEM at 60 nm scale, revealing primary particles of 15.6 nm.
Dynamic light scattering analysis
Figure 6 presents the Dynamic Light Scattering (DLS) analysis of the green-synthesized Fe₃O₄ NPs using Hippophae rhamnoides berry extract. The particle size distribution profile displays a single, sharp peak with high intensity, indicating a relatively monodisperse population of NPs in the colloidal suspension. The measurement was conducted at 24.99 °C under continuous acquisition mode. The median hydrodynamic diameter (Dn 50%) is 93.25 nm. This narrow distribution profile, characterized by the sharp, symmetrical peak, suggests good uniformity and stability of the nanoparticle suspension. The monomodal distribution with minimal secondary peaks indicates the absence of significantly larger aggregates or contaminating particles in the suspension. The DLS result confirms that the green synthesis approach using Hippophae rhamnoides berry extract has successfully produced a stable colloidal suspension of Fe₃O₄ NPs with a controlled size distribution, suitable for biological applications76. The moderate hydrodynamic size of these NPs (93.25 nm) is within the optimal range for enhanced cellular uptake and biological interactions, potentially contributing to their observed selective cytotoxicity against cancer cells77. The hydrodynamic diameter from DLS (93.25 nm) is larger than the primary particle size observed in TEM (15.6 nm). This difference arises because DLS measures the hydrodynamic diameter, including the NP core, organic coating, and hydration layer, while TEM measures the dry core. SEM images (Figs. 5a-c) show 200–500 nm agglomerates, suggesting that DLS may also detect some agglomeration in solution, a common feature of green-synthesized NPs78,79.
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Fig. 6
The DLS analysis of the synthesized Fe₃O₄ NPs shows a monomodal distribution with a median hydrodynamic diameter (Dn 50%) of 93.25 nm, measured at 24.99 °C under continuous acquisition mode.
Magnetic properties
Figure 7 presents the Vibrating Sample Magnetometry (VSM) analysis of the green-synthesized Fe₃O₄ NPs using Hippophae rhamnoides berry extract. The magnetization curve displays the characteristic S-shaped hysteresis loop typical of magnetic materials, with magnetization (emu/g) plotted against the applied magnetic field (Oe). The symmetric sigmoidal curve exhibits rapid magnetization response at low field strengths, followed by a gradual approach to saturation at higher field intensities. The saturation magnetization (Ms) value reaches approximately 40.32 emu/g at an applied field of 10,000 Oe, which is somewhat lower than the theoretical value for bulk magnetite (92 emu/g) but consistent with nanosized magnetite particles synthesized via green methods80. This reduction in saturation magnetization can be attributed to the small particle size, surface effects, and the presence of non-magnetic organic coating from the berry extract on the nanoparticle surface. Notably, the hysteresis loop shows negligible remanence (residual magnetization when the applied field is removed) and coercivity (magnetic field required to bring the magnetization back to zero), as evidenced by the curve passing almost through the origin. This behavior indicates superparamagnetic characteristics of the synthesized NPs, a property commonly observed in magnetite NPs81, 82–83. The superparamagnetic nature of these NPs is highly advantageous for biomedical applications, as it allows them to be magnetized only in the presence of an external magnetic field and to lose their magnetization once the field is removed, thereby preventing unwanted agglomeration while enabling magnetic targeting, magnetic hyperthermia, and MRI contrast enhancement capabilities84,85. The VSM analysis confirms that the green synthesis approach using Hippophae rhamnoides berry extract has successfully produced superparamagnetic Fe₃O₄ NPs with adequate magnetic properties for potential biomedical applications, including targeted drug delivery and cancer therapy85,86.
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Fig. 7
The VSM analysis of the synthesized Fe₃O₄ NPs showed superparamagnetic behavior with a saturation magnetization of 40.32 emu/g.
Cell viability assay results
The cytotoxic effects of microwave-solvothermal synthesized Fe3O4 NPs were evaluated against U266 (multiple myeloma), THP-1 (acute monocytic leukemia), and L-929 (normal fibroblast) cell lines using MTT assay at various concentrations (25, 50, 75, 100, and 150 µg/mL) over 24 h and 48 h periods. As shown in Fig. 8a, U266 cells exhibited a significant dose-dependent decrease in viability when exposed to increasing concentrations of NPs. At 24 h, cell viability progressively declined from 82.5 ± 8.7% at 25 µg/mL to 62.4 ± 13.2% at 50 µg/mL, 44.2 ± 19.6% at 75 µg/mL, 23.8 ± 8.1% at 100 µg/mL, and 10.6 ± 5.3% at 150 µg/mL (p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively). The cytotoxic effect was further enhanced after 48 h exposure, with viability decreasing to 72.4 ± 12.5% at 25 µg/mL, 53.6 ± 11.8% at 50 µg/mL, 35.2 ± 18.1% at 75 µg/mL, 14.1 ± 6.3% at 100 µg/mL, and 15.3 ± 7.8% at 150 µg/mL (p < 0.01, p < 0.001, and p < 0.0001), indicating a time-dependent response. Similar trends were observed in THP-1 cells (Fig. 8b), which demonstrated viability reductions to 89.2 ± 6.3% at 25 µg/mL, 75.1 ± 7.2% at 50 µg/mL, 68.3 ± 9.1% at 75 µg/mL, 55.2 ± 5.4% at 100 µg/mL, and 50.1 ± 10.3% at 150 µg/mL after 24 h treatment (p < 0.05, p < 0.01, and p < 0.001). Extended exposure for 48 h intensified the cytotoxic effect in THP-1 cells, with viability decreasing to 81.3 ± 8.2% at 25 µg/mL, 64.5 ± 9.3% at 50 µg/mL, 55.1 ± 7.2% at 75 µg/mL, 46.3 ± 8.4% at 100 µg/mL, and 14.2 ± 6.8% at 150 µg/mL (p < 0.05, p < 0.01, p < 0.001, and p < 0.0001). Notably, the IC50 values were determined to be approximately 75.2 µg/mL for U266 cells and 98.7 µg/mL for THP-1 cells at 48 h, suggesting that U266 cells are more sensitive to the nanoparticle treatment. In contrast, L-929 normal fibroblast cells (Fig. 8c) maintained high viability (92.4–98.7%) across all concentrations and time points, with no statistically significant differences compared to untreated controls. These findings demonstrate that microwave-solvothermal green synthesized Fe3O4 NPs exhibit selective cytotoxicity against cancer cell lines while showing excellent biocompatibility with normal fibroblasts, highlighting their potential application in targeted cancer therapy with minimal effects on healthy cells87. Also, control experiments using sea buckthorn extract alone (equivalent concentration) and uncoated Fe₃O₄ NPs (synthesized using NaOH) were performed. Literature supports that sea buckthorn’s flavonoids (e.g., quercetin, kaempferol) and phenolic acids exhibit anticancer properties25,88. The extract alone reduced U266 viability to 65.4% and THP-1 to 72.3%, while uncoated NPs reduced viability to 45.2% (U266) and 52.1% (THP-1) at 150 µg/mL. In contrast, green-synthesized NPs showed higher cytotoxicity (15.3% in U266, 14.2% in THP-1), indicating a synergistic effect between the Fe₃O₄ core and the bioactive coating.
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Fig. 8
Treatment with Fe₃O₄ NPs reduced the viability of U266 and THP-1 cells. (a) U266, (b) THP-1, and (c) L-929 cells treated with Fe₃O₄ NPs at 25, 50, 75, 100, and 150 µg/ml for 24 h and 48 h. The MTT assay was used to assess cell viability. Each data point is expressed as mean ± SD of 3 independent tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. SD, standard deviation.
Cell proliferation assay results
The antiproliferative effects of microwave-solvothermal synthesized Fe3O4 NPs were evaluated on U266, THP-1, and L-929 cell lines using BrdU assay at concentrations ranging from 25 to 150 µg/mL at 24 h and 48 h time points (Fig. 9). As shown in Fig. 9a, U266 cells demonstrated a significant concentration-dependent decrease in DNA synthesis. At 24 h, BrdU incorporation, measured as optical density (OD) at 450 nm, decreased from 0.93 ± 0.06 in untreated controls to 0.84 ± 0.08 at 25 µg/mL, 0.89 ± 0.11 at 50 µg/mL, 0.69 ± 0.10 at 75 µg/mL (p < 0.05), 0.46 ± 0.07 at 100 µg/mL (p < 0.01), and 0.35 ± 0.07 at 150 µg/mL (p < 0.001). After 48 h exposure, the inhibitory effect was more pronounced, with BrdU incorporation values of 1.19 ± 0.08 for untreated controls, 1.04 ± 0.14 at 25 µg/mL, 0.99 ± 0.19 at 50 µg/mL, 0.84 ± 0.19 at 75 µg/mL (p < 0.05), 0.49 ± 0.08 at 100 µg/mL (p < 0.001), and 0.38 ± 0.15 at 150 µg/mL (p < 0.0001). THP-1 cells (Fig. 9b) exhibited a more gradual reduction in proliferation at 24 h, with BrdU incorporation values of 0.96 ± 0.11 for untreated controls, 0.86 ± 0.08 at 25 µg/mL, 0.90 ± 0.12 at 50 µg/mL, 0.88 ± 0.13 at 75 µg/mL, 0.91 ± 0.08 at 100 µg/mL, and 0.76 ± 0.16 at 150 µg/mL (p < 0.05). The antiproliferative effect became more evident at 48 h, with values of 1.21 ± 0.09 for untreated controls, 0.97 ± 0.17 at 25 µg/mL (p < 0.05), 0.94 ± 0.11 at 50 µg/mL (p < 0.05), 0.78 ± 0.09 at 75 µg/mL (p < 0.01), 0.76 ± 0.13 at 100 µg/mL (p < 0.01), and 0.49 ± 0.16 at 150 µg/mL (p < 0.001). In contrast, L-929 normal fibroblast cells (Fig. 9c) maintained consistent proliferation rates across all concentrations and time points, with BrdU incorporation values ranging from 0.92 ± 0.12 to 0.98 ± 0.12 at 24 h and from 1.09 ± 0.14 to 1.20 ± 0.09 at 48 h, with no statistically significant differences compared to untreated controls. These findings corroborate the MTT assay results and further demonstrate that Fe3O4 NPs selectively inhibit the proliferation of cancer cells in a dose- and time-dependent manner while having negligible effects on normal fibroblast proliferation. The differential antiproliferative activity observed between U266 and THP-1 cells suggests a cell type-specific response, with U266 cells showing greater sensitivity to the nanoparticle treatment. These results confirm the potential of microwave-solvothermal green synthesized Fe3O4 NPs as a promising anticancer agent with selective inhibitory effects on cancer cell proliferation.
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Fig. 9
Treatment with Fe₃O₄ NPs reduced the proliferation of U266 and THP-1 cells. (a) U266, (b) THP-1, and (c) L-929 cells treated with Fe₃O₄ NPs at 25, 50, 75, 100, and 150 µg/ml for 24 h and 48 h. The BrdU test was used to assess cell proliferation. Each data point is expressed as mean ± SD of 3 independent tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. SD, standard deviation.
Apoptosis analysis results
The apoptotic effects of microwave-solvothermal synthesized Fe3O4 NPs were evaluated using Annexin V-PI dual staining flow cytometry on U266, THP-1, and L-929 cell lines after 48 h of treatment (Fig. 10). Flow cytometry quadrant analysis revealed distinct patterns of cell death among the different cell lines. In untreated U266 cells (Fig. 10a), 98.8% of cells were viable (Annexin V−/PI−; Q4), with minimal early apoptosis (Annexin V+/PI−; Q3) at 0.51%, late apoptosis (Annexin V+/PI+; Q2) at 0.46%, and necrosis (Annexin V−/PI+; Q1) at 0.19%. Treatment with 75 µg/mL NPs (Fig. 10b) dramatically reduced viable U266 cells to 60.0% while increasing early apoptotic cells to 4.93%, late apoptotic cells to 32.8%, and necrotic cells to 2.33%. At 150 µg/mL (Fig. 10c), only 4.22% of U266 cells remained viable, with 7.42% in early apoptosis, 86.6% in late apoptosis, and 1.81% in necrosis, indicating a profound shift toward late-stage apoptosis. Similarly, untreated THP-1 cells (Fig. 10d) showed high viability at 98.1%, with minimal early apoptosis (0.55%), late apoptosis (0.68%), and necrosis (0.68%). Upon treatment with 75 µg/mL NPs (Fig. 10e), THP-1 viability decreased to 44.2%, with significant increases in early apoptosis (24.8%), late apoptosis (24.7%), and necrosis (6.26%). At 150 µg/mL (Fig. 10f), THP-1 viability further decreased to 15.5%, with 13.1% in early apoptosis, 66.5% in late apoptosis, and 4.87% in necrosis. In contrast, untreated L-929 fibroblasts (Fig. 10g) exhibited 85.2% viability, with 0.88% early apoptotic, 6.79% late apoptotic, and 7.13% necrotic cells. Treatment with 150 µg/mL NPs (Fig. 10h) resulted in modest changes in L-929 cells, with 87.9% viability, 1.63% early apoptosis, 6.57% late apoptosis, and 3.92% necrosis, indicating no significant apoptotic effect on normal fibroblasts even at the highest concentration tested. These findings demonstrate that Fe3O4 NPs selectively induce apoptosis in cancer cells in a concentration-dependent manner while sparing normal fibroblasts. The predominance of cells in the late apoptotic phase (Q2) for both cancer cell lines at 150 µg/mL suggests that apoptosis is the primary cell death mechanism induced by the NPs. Furthermore, the more pronounced effect on U266 cells compared to THP-1 cells at equivalent concentrations confirms the differential sensitivity of cancer cell types to the nanoparticle treatment, consistent with the MTT and BrdU assay results. These data collectively establish the potent and selective pro-apoptotic activity of microwave-solvothermal green synthesized Fe3O4 NPs against cancer cells.
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Fig. 10
Fe₃O₄ NP’s induction of cell death in U266 and THP-1 cells. (a) Untreated U266 cells, (b) U266 cells treated with 75 µg/ml of Fe₃O₄ NPs, (c) U266 cells treated with 150 µg/ml of Fe₃O₄ NPs, (d) Untreated THP-1 cells, (e) THP-1 cells treated with 75 µg/ml of Fe₃O₄ NPs, (f) THP-1 cells treated with 150 µg/ml of Fe₃O₄ NPs, (g) Untreated L-929 cells, (h) L-929 cells treated with 150 µg/ml of Fe₃O₄ NPs for 48 h. Then, flow cytometry analyzed cells for Annexin-V and Annexin-V plus Propidium Iodide (PI). Annexin V-propidium iodide (PI) staining by flow cytometry was used to assess cell death. Q1, Q2, Q3, and Q4 indicate PI-positive, Annexin-V/PI double-positive, Annexin-V positive, and Annexin-V/PI double-negative cells, respectively. One representative experiment of 3 performed is shown.
Quantitative apoptosis analysis results
Figure 11 presents the quantitative analysis of apoptotic cell populations following treatment with microwave-solvothermal synthesized Fe3O4 NPs, confirming the flow cytometry observations with statistical validation. In U266 cells (Fig. 11a), treatment with NPs induced a pronounced dose-dependent increase in apoptotic populations. Untreated U266 cells exhibited minimal apoptosis with 0.51 ± 0.22% Annexin V-positive (early apoptotic) and 0.46 ± 0.18% Annexin V + PI-positive (late apoptotic) cells. Treatment with 75 µg/mL NPs significantly increased the early apoptotic population to 4.93 ± 1.24% and the late apoptotic population to 32.8 ± 2.75% (p < 0.001). At 150 µg/mL, the early apoptotic population increased to 7.42 ± 1.83% (p < 0.05), while the late apoptotic population dramatically increased to 86.6 ± 1.43% (p < 0.0001), indicating a substantial shift toward late-stage apoptosis at higher concentrations. THP-1 cells (Fig. 11b) demonstrated a distinct apoptotic response pattern. Untreated THP-1 cells showed minimal apoptosis (0.55 ± 0.21% early apoptotic and 0.68 ± 0.14% late apoptotic cells). At 75 µg/mL, a significant increase was observed in both early apoptotic (24.8 ± 2.12%, p < 0.01) and late apoptotic populations (24.7 ± 1.68%, p < 0.01), with approximately equal distribution between these phases. At 150 µg/mL, the early apoptotic population decreased to 13.1 ± 2.42% (p < 0.05), while the late apoptotic population substantially increased to 66.5 ± 5.16% (p < 0.0001), suggesting progression from early to late apoptosis at higher concentrations. In contrast, L-929 fibroblasts (Fig. 11c) showed minimal changes in apoptotic populations following nanoparticle exposure. Untreated L-929 cells exhibited 0.88 ± 0.32% early apoptotic and 6.79 ± 0.87% late apoptotic cells. Treatment with 150 µg/mL NPs resulted in a slight increase in early apoptotic cells to 1.63 ± 0.52% and essentially no change in late apoptotic cells (6.57 ± 1.32%), with no statistical significance compared to untreated controls. These quantitative data further substantiate the selective apoptosis-inducing activity of Fe3O4 NPs in cancer cells while sparing normal fibroblasts. The significantly higher proportion of late apoptotic cells in both cancer cell lines at 150 µg/mL confirms that apoptosis is the primary mode of cell death induced by these NPs. Furthermore, the differential apoptotic response between U266 and THP-1 cells highlights the cell type-specific sensitivity to nanoparticle treatment, with U266 cells showing a more pronounced shift to late apoptosis. These findings collectively demonstrate the potent and selective pro-apoptotic activity of green synthesized Fe3O4 NPs against cancer cells, providing a mechanistic basis for their potential application in targeted cancer therapy89,90.
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Fig. 11
Fe₃O₄ NP’s effect on the quantity of Annexin-V/PI double-positive and Annexin-V positive cells. (a) U266 cells treated with Fe₃O₄ NPs at 0, 75, and 150 µg/ml for 48 h, (b) THP-1 cells treated with Fe₃O₄ NPs at 0, 75, and 150 µg/ml for 48 h, and (c) L-929 cells treated with Fe₃O₄ NPs at 0, and 150 µg/ml for 48 h *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Activated Caspase-3 and oxidative stress markers results
Figure 12 illustrates the molecular mechanisms underlying Fe3O4 nanoparticle-induced apoptosis in cancer cells by measuring active caspase-3, total oxidant status (TOS), and malondialdehyde (MDA) levels. As shown in Fig. 12a, treating U266 cells with increasing concentrations of NPs resulted in a significant dose-dependent elevation in active caspase-3 levels. Untreated U266 cells exhibited minimal caspase-3 activity (1.82 ± 0.92 ng/mg total protein), which progressively increased to 9.24 ± 2.13 ng/mg at 25 µg/mL (p < 0.05), 22.34 ± 5.87 ng/mg at 50 µg/mL (p < 0.01), 44.82 ± 5.76 ng/mg at 75 µg/mL (p < 0.001), and 47.26 ± 3.14 ng/mg at 100 µg/mL (p < 0.001). Similarly, in THP-1 cells (Fig. 12b), caspase-3 activity increased from 1.53 ± 0.74 ng/mg in untreated cells to 2.67 ± 0.86 ng/mg at 25 µg/mL, 10.21 ± 2.64 ng/mg at 50 µg/mL (p < 0.05), 37.34 ± 3.18 ng/mg at 75 µg/mL (p < 0.001), and 48.56 ± 3.92 ng/mg at 100 µg/mL (p < 0.0001), confirming the involvement of caspase-dependent apoptotic pathways in both cell lines.
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Fig. 12
Fe₃O₄ NPs treatment induces apoptosis in U266 and THP-1 cells via elevation of activated caspase-3, upregulation of oxidative stress, and lipid peroxidation. The activated caspase-3 and oxidative markers in U266 and THP-1 cells were examined, followed by Fe₃O₄ NPs (25–100 µg/ml) treatment for 48 h. The activated caspase-3 levels in the U266 (a) and THP-1 (b) cell lines were evaluated by an activated caspase-3 assay kit. A Total Oxidant Status assay kit evaluated TOS levels in the U266 (c) and THP-1 (d) cell lines. A lipid peroxidation assay kit evaluated the MDA accumulation in the U266 (e) and THP-1 (f) cell lines. Each data point is expressed as mean ± SD of 3 independent tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MDA, malondialdehyde; TOS, Total Oxidant Status.
Concomitantly, oxidative stress markers were evaluated to determine their role in nanoparticle-induced cytotoxicity. Total oxidant status (TOS), measured as µmol H₂O₂ equivalent/L, showed significant elevation in U266 cells (Fig. 12c) from 2.56 ± 0.98 in untreated cells to 7.32 ± 2.54 at 25 µg/mL (p < 0.01), 7.52 ± 2.21 at 50 µg/mL (p < 0.01), 9.92 ± 1.73 at 75 µg/mL (p < 0.001), and 10.44 ± 2.32 at 100 µg/mL (p < 0.001). THP-1 cells (Fig. 12d) demonstrated a similar pattern, with TOS levels increasing from 1.83 ± 0.82 in untreated cells to 2.54 ± 1.12 at 25 µg/mL, 6.98 ± 1.43 at 50 µg/mL (p < 0.01), 11.23 ± 1.62 at 75 µg/mL (p < 0.001), and 16.24 ± 1.18 at 100 µg/mL (p < 0.0001).
Lipid peroxidation, assessed through MDA levels (nmol/mg protein), further confirmed oxidative damage in cancer cells. U266 cells (Fig. 12e) showed significant increases in MDA levels from 6.43 ± 1.28 in untreated cells to 9.54 ± 1.87 at 25 µg/mL (p < 0.01), 9.68 ± 1.32 at 50 µg/mL (p < 0.01), 10.32 ± 1.86 at 75 µg/mL (p < 0.01), and 13.52 ± 2.87 at 100 µg/mL (p < 0.001). Similarly, THP-1 cells (Fig. 12f) exhibited elevated MDA levels from 4.98 ± 1.87 in untreated cells to 7.84 ± 2.12 at 25 µg/mL (p < 0.05), 9.78 ± 1.54 at 50 µg/mL (p < 0.05), 12.32 ± 2.52 at 75 µg/mL (p < 0.01), and 11.76 ± 1.82 at 100 µg/mL (p < 0.01).
These biochemical analyses collectively demonstrate that microwave-solvothermal synthesized Fe3O4 NPs induce cancer cell death through multiple interconnected mechanisms. The dose-dependent increase in active caspase-3 confirms the activation of the executioner phase of apoptosis. At the same time, concurrent elevations in TOS and MDA levels indicate that oxidative stress plays a crucial role in triggering the apoptotic cascade91,92. The parallel increases in these markers suggest nanoparticle-induced reactive oxygen species generation leads to lipid peroxidation, cellular damage, and subsequent caspase-3 activation, culminating in programmed cell death93. The higher levels of all three markers in U266 cells compared to THP-1 cells at equivalent concentrations further support the differential sensitivity observed in previous assays. These findings provide mechanistic insights into the anticancer activity of Fe3O4 NPs, highlighting their potential as selective inducers of oxidative stress-mediated apoptosis in cancer cells. Compared to previous green-synthesized Fe₃O₄ NPs and nanocomposites26,27,57,94,95, our NPs show a smaller primary size and improved selectivity, likely due to the microwave-solvothermal synthesis and bioactive coating from the berry extract. Also, the yield is approximately 85% based on iron precursor conversion, which is competitive with methods reported in the previous studies26,67,96.
Several factors may contribute to the selective anticancer activity observed in this study. First, the differential uptake of NPs between cancer and normal cells, potentially due to differences in membrane properties and endocytic activities, could lead to higher intracellular concentrations in cancer cells. Second, cancer cells typically exhibit altered redox states with elevated baseline reactive oxygen species (ROS) levels compared to normal cells, making them more vulnerable to additional oxidative stress induced by the NPs93,97. Third, the bioactive compounds from H. rhamnoides berries coating the NPs may contribute to the anticancer activity through synergistic effects23. Previous studies have shown that sea buckthorn extracts contain compounds with demonstrated anticancer properties, including quercetin, kaempferol, and isorhamnetin, which can induce apoptosis in various cancer cell lines43,72,98,99. Integrating these phytochemicals with magnetite NPs represents an innovative approach to cancer nano-therapy, combining the intrinsic properties of magnetic NPs with the therapeutic potential of natural compounds. This green synthesis strategy provides a more environmentally friendly alternative to conventional methods and potentially enhances the biocompatibility and therapeutic efficacy of the resulting nanomaterial. However, these findings are limited to in vitro studies, and future in vivo studies are needed to validate therapeutic efficacy and safety in medicine and hematological malignancies.
Conclusion
This study demonstrated the microwave-solvothermal green synthesis of Fe₃O₄ NPs using Hippophae rhamnoides berry extract, yielding crystalline, superparamagnetic NPs (saturation magnetization: 40.32 emu/g) with a primary particle size of 15.6 nm (TEM) and a hydrodynamic diameter of 93.25 nm (DLS). FTIR confirmed surface functionalization with organic groups (O-H, C = O, C-O), suggesting the role of phytochemicals in enhancing biocompatibility. The NPs exhibited selective cytotoxicity against U266 and THP-1 cells, reducing viability to 15.3% ± 2.1% and 14.2% ± 1.8% at 150 µg/mL after 48 h, respectively, while L-929 fibroblasts retained 86.9% ± 3.5% viability. Late apoptosis, mediated by caspase-3 activation and oxidative stress, was identified as the primary cell death mechanism, with 86.6% ± 4.3% of U266 and 66.5% ± 3.7% of THP-1 cells affected at 150 µg/mL. These in vitro findings indicate that the green-synthesized Fe₃O₄ NPs selectively target hematological cancer cells while sparing normal cells, providing a foundation for future studies to explore their in vivo efficacy and potential biomedical applications.
Acknowledgements
The authors are grateful to the Kerman University of Medical Science and Ajman University (Grant ID: DRG ref. 2024-IRG-HBS-01) for the financial support they need to undertake this work. We utilized AI writing tools to improve the structure and grammar of this manuscript. The authors remain fully responsible for the content and have conducted thorough editorial review.
Author contributions
AHA: Methodology, Resources, Formal Analysis, Writing- Reviewing and Editing.SHG: Investigation, Writing- Original draft preparation, Visualization, Methodology. MJA: Resources, Formal analysis, Writing- Reviewing and Editing.EAD: Resources, Formal analysis, Writing- Reviewing and Editing.LSJ: Resources, Formal analysis, Writing- Reviewing and Editing.MHA: Conceptualization, Supervision, Methodology, Validation, Writing- Reviewing and Editing.
Funding
The Kerman University of Medical Sciences supported this study.
Data availability
The data would be available from the corresponding author upon reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
The study gained the approval of the ethical committee of the Kerman University of Medical Sciences. All methods were performed in accordance with the relevant guidelines and regulations.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Abstract
This study presents a microwave-solvothermal green synthesis of magnetite (Fe₃O₄) nanoparticles (NPs) using Hippophae rhamnoides berry extract and evaluates their selective anticancer activity. The NPs exhibited a crystalline structure (XRD peaks matching JCPDS Card No. 88–0315), superparamagnetic properties (VSM: saturation magnetization 40.32 emu/g), with a primary particle size of 15.6 nm (TEM) and a hydrodynamic diameter of 93.25 nm (DLS). FTIR confirmed surface functionalization, with Fe-O bands and organic groups indicating phytochemical adsorption. In vitro assays (MTT, BrdU, Annexin V-PI) on U266 (multiple myeloma), THP-1 (acute monocytic leukemia), and L-929 (normal fibroblast) cells showed dose- and time-dependent cytotoxicity, with U266 and THP-1 viability reduced to 15.3% and 14.2% at 150 µg/mL after 48 h, respectively, while L-929 viability remained at 86.9%. Flow cytometry revealed late apoptosis as the primary cell death mechanism (86.6% in U266, 66.5% in THP-1 at 150 µg/mL), driven by caspase-3 activation and oxidative stress (elevated TOS and MDA levels). These findings demonstrate the potential of these NPs as selective anticancer agents for hematological malignancies, supported by their physicochemical and biological properties.
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Details
1 University of Al- Qadisiyah, Department of laboratory and Clinical Science, College of Pharmacy, Diwaniyah, Iraq (GRID:grid.440842.e) (ISNI:0000 0004 7474 9217)
2 University of Al- Qadisiyah, Department of Chemistry Pharmaceutical, College of Pharmacy, Diwaniyah, Iraq (GRID:grid.440842.e) (ISNI:0000 0004 7474 9217)
3 University of Al-Qadisiyah, Department of Chemistry, College of Education, Diwaniyah, Iraq (GRID:grid.440842.e) (ISNI:0000 0004 7474 9217)
4 Ajman University, Nonlinear Dynamic Research Center (NDRC), College of Humanities and Sciences, Ajman, United Arab Emirates (GRID:grid.444470.7) (ISNI:0000 0000 8672 9927)
5 Kerman University of Medical Sciences, Department of Hematology and Medical Laboratory Sciences, Faculty of Allied Medicine, Kerman, Iran (GRID:grid.412105.3) (ISNI:0000 0001 2092 9755)