Introduction

Acute myeloid leukemia (AML) is the most commonly diagnosed form of acute leukemia in older individuals. Enhanced comprehension of the biology and genetic structure of AML has resulted in improved evaluation of the risk associated with this disease. Although there have been some encouraging recent developments, the mainstay of treatment still involves using a mix of cytarabine- and anthracycline-based treatment plans, along with allogeneic stem cell transplantation for suitable patients. Despite the implementation of many treatments, there has been no substantial improvement in the overall survival or disease-free survival rates of patients in clinical settings. Consequently, the prognosis for patients with AML is frequently characterized as extremely unfavorable or unsatisfactory.1 The efficacy of various chemotherapeutic agents against AML has been hindered by several factors: (1) the narrow therapeutic window of these drugs, (2) the development of drug resistance in refractory AML, (3) inadequate impact on dormant cells like leukemic stem cells, and (4) unintended effects on non-target tissues. Therefore, there is still a need for the development of novel and targeted therapeutic approaches that can address the limitations of conventional chemotherapy.2

The emergence of nanoscale technology marks a significant development in the realm of cancer therapy. Due to their distinct physical and biological characteristics, nanotechnology-based medications have been included into cancer treatment and have significantly transformed the conventional methods of cancer therapy.3 There has been a significant increase in research focused on designing and creating nanoparticle systems. These systems have resulted in the development of diverse material formulations that show promise therapeutic effects for treating different forms of cancer. Nanoparticles (NPs) are structures ranging in size from 1 to 100 nm, characterized by a significantly high surface-to-volume ratio. This unique feature gives rise to distinct behaviors not observed in larger particles of the same chemical makeup. The NPs are utilized in several biological applications due to their distinctive characteristics and their ability to penetrate cells and organelles owing to their nanoscale size.4 Specifically, researchers have extensively studied inorganic NPs for their medicinal potential in treating malignant disorders. Inorganic NPs exhibit distinct physicochemical characteristics, including facile manufacturing, a high ratio of surface area to volume, stability, and the potential for surface modification to enhance their affinity and selectivity towards specific molecules.5 Metallic nanoparticles such as Nobel metals like Au, Pt, Pd and Ag) are being used in different biomedical, applications due to their significant physicochemical properties. However, several investigations have listed their side effect on human. The cytotoxicity of silver NP (AgNPs) even at a low limit of exposure dosage (10–50 μg/mL) was reported in the literature,6 whereas iron nanoparticles (FeNPs) at the level (100 μg/mL) showed low or no cytotoxicity as reported in previous report.7 It was reported in the literature that; the iron based nanoparticles (FeNPs) are considered using in several sectors by FDA (US Food and Drug Administration), including magnetic resonance imaging for liver pathologies detection, iron deficiency anemia therapy in adults with chronic kidney disease.8–11

The biocompatible and biodegradable nature of iron oxide NPs, along with their easy manufacturing and capacity to be tailored and functionalized for specific purposes, make them highly promising for applications in medical cancer. Concurrently with the extensive range of their biological uses, there has been a rise in safety concerns regarding iron oxide NPs. Numerous investigations on the cytotoxicity of iron oxide NPs have been conducted. Iron oxide NPs are widely believed to have minimal or no harmful effects on cells. Nevertheless, studies have demonstrated that iron oxide NPs exhibit various harmful effects on cells, and diverse types of cells have displayed considerable vulnerability to the cytotoxicity of iron oxide NPs.12

Carbon nanotubes (CNTs) are a diverse group of nanomaterials that have a wide range of physical, chemical, mechanical, and biological properties. Due to their exceptional properties, CNTs have garnered significant interest in the field of biomedicine, namely in the realm of cancer treatment.13 The extensive utilization of CNTs has prompted significant apprehensions regarding their safety. Despite several researches undertaken to assess the cytotoxicity of CNTs, the available data sometimes present conflicting results. Several studies have documented that CNTs can cause cytotoxicity in various cell lines, with the extent of toxicity being depending on both the duration and concentration of exposure. Nevertheless, alternative studies indicate the absence of cytotoxicity after administering CNTs.14

Carbon nanoparticles possess a significant surface area, which enables the potential for surface functionalization. Carbon nanomaterials are highly suitable for the attachment of NPs. Lately, there has been significant interest in the bonding of metal NPs to carbon nanomaterials. Metal NPs incorporated with carbon nanomaterials form nanohybrids that possess not only the distinct characteristics of the metal NPs and carbon nanomaterials, but also demonstrate improved features resulting from the interactions between these two components. The integration of metal NPs with carbon nanomaterials produces innovative and adaptable nanosystems that have significant potential in the field of biomedicine. Specifically, these nanosystems offer new possibilities for the development of cancer therapies based on nanotechnology.15

The uptake and behavior of nanosystems in biological environments are significantly influenced by three important parameters: particle size, coating, and stability. Typically, the iron oxide NPs rapidly clump together under conditions of normal body pH and in the presence of ions. Furthermore, the limited stability and varied size distribution in water are significant drawbacks of iron oxide NPs. To mitigate these drawbacks, the surface of iron oxide NPs can be coated with different polymers, particularly biopolymers like polysaccharides. This coating process results in the production of uniform, stable, and biocompatible iron oxide NPs.16 The CNTs exhibit a propensity to congregate, resulting in their insolubility. In order to decrease the aggregation, the CNTs might be covered with synthetic or natural polymers.17 Gum Arabic (GA) is a naturally-occurring, non-toxic, water-loving, extensively-branched polysaccharide obtained from the exudates of Acacia senegal and Acacia seyal trees. Due to its biocompatibility and its ability to stabilize and emulsify, GA has been applied in the field of nanomedicine.18

Although many studies have been conducted to investigate the biological effects of magnetite (Fe3O4) NPs and CNTs, the cytotoxic effects of Fe3O4 NPs and CNTs remain contradictory. The current knowledge is inconclusive, with numerous contrasting outcomes. While several studies have indicated minimal or absent cytotoxicity, others have reported notable cytotoxic effects. Furthermore, the complete understanding of the possible lethal effects and the underlying molecular pathways of Fe3O4 NPs and CNTs on AML cells is still lacking. To reduce the ambiguity surrounding the toxicity of Fe3O4 NPs and CNTs, and to establish definitive evidence of their harmful effects on AML cells, we conducted a comprehensive assessment of the cytotoxic properties of GA-stabilized/destabilized Fe3O4 NPs and CNTs, both individually and in combination, on AML cell lines. Furthermore, the fundamental molecular pathways responsible for the cytotoxic effects of nanostructures were also examined.

Materials and Methods Materials

Multi-walled CNTs (Lot No. mst-200) were purchased from Nanotech Co., Ltd. (South Korea). The CNTs had a length ranging from 10 to 50 μm, a diameter ranging from 15 to 30 nm, a Brunauer–Emmett–Teller (BET) surface area of around 200 m2/g, and a purity of 95%. The GA was obtained from exudates of Acacia senegal trees sourced from several regions of Saudi Arabia. All reagents utilized in nanostructures preparation and characterization, specifically ferric chloride hexahydrate, ferrous sulfate heptahydrate, ammonia solution, sulfuric acid, and nitric acid, are of laboratory grade.

Methods Purification of GA and CNTs

A solution containing GA was created and purified to aid in stabilizing the CNTs, generated Fe3O4 NPs, and Fe3O4/CNTs nanocomposite. A stabilizing solution was made by boiling 1 gram of extracted GA powder in 100 milliliters of distilled water with continuous magnetic stirring until a gelatinous solution formed. The solution was filtered to eliminate any suspended contaminants, subsequently cooled, and stored at room temperature. As for CNTs, the unprocessed powder of the CNTs underwent a purification procedure that included subjecting it to heat treatments at a temperature of 450°C for a duration of 30 minutes. This process aimed to eliminate any organic binder that may potentially contaminate the surface of the powder. In addition, the specimen underwent multiple rinses using acetone to eliminate any extraneous organic contaminants. The CNTs powder generated after the heat treatment procedure underwent acid functionalization using ultrasonication at a frequency of 40 kHz and a temperature of 60°C in a combination of H2SO4 and HNO3 for a duration of 15 minutes. This process was carried out to introduce functional groups onto the surface of the CNTs. The acquired CNTs were thoroughly rinsed with distilled water to eliminate any residual acids.

Synthesis and Stabilization of Fe3O4/CNTs Nano-Hybrid Composite

A calculated quantity of the purified CNTs was subjected to ultrasonication at a frequency of 40 kHz using the prepared solution of GA. This GA solution was employed to create a suspension of CNTs-GA hybrid. The Co-precipitation technique was used to prepare Fe3O4 NPs and Fe3O4/CNTs nanocomposites, which were stabilized with GA. Concisely, a solution containing 4 millimoles of ferric chloride hexahydrate and another solution containing 2 millimoles of ferrous sulphate heptahydrate were subjected to sonication at room temperature, with and without the calculated amounts of the CNTs to prepare Fe3O4/CNTs nanocomposite and Fe3O4 NPs, respectively. Ammonia solution (NH4OH) is incrementally added to the previous solution until the co-precipitation process is finished. The pH of the solution is adjusted by using a pH glass electrode. Subsequently, the GA solution was introduced into the aforementioned combination while subjecting it to sonication for a duration of 15 minutes, resulting in the formation of the Fe3O4-GA and Fe3O4/CNTs-GA hybrid solution. Figure 1 depicts a schematic flowchart detailing the step-by-step co-precipitation synthesis process of the studied NPs. The volume fraction (Vf) of CNTs in the Fe3O4/CNTs nanocomposite particles was determined using a simplified approximation based on the theoretical value of CNTs volume fraction, following the rule of mixtures. The analysis revealed that the calculated volume fraction for the synthesized 1 wt % Fe3O4/CNTs is around 5 Vol. % Fe3O4/CNTs, with Fe3O4 having a density of 5.17 g/cm3 and CNTs having a density of 1.74 g/cm3.

Figure 1 Experimental procedure design of GA-stabilized Fe3O4 and Fe3O4/CNTs nano-hybrid composite particles by co-precipitation process.

Characterization and Investigations of NPs

The particle morphology, shape, and size of CNTs, as well as the produced Fe3O4 NPs and the Fe3O4/CNTs nanocomposites, were examined using a Field emission scanning electron microscope (FE-SEM) equipped with an energy-dispersive X-ray (EDS) unit (JEOL, JSM-7600F). A transmission electron microscope (TEM) of the model JEOL, JEM-1011 operated at an acceleration voltage of 300 kV was used to investigate the size, shape and surface morphology of the used CNTs. The X-ray diffractometer (XRD) (D8 DISCOVER, Bruker, MA, USA) was used to analyze the crystal structure and chemical content of the generated nanoparticles (NPs). The FTIR spectra of the produced NPs was measured using an FTIR-4600 Fourier Transform Infrared spectrometer manufactured by JASCO Corporation in Tokyo, Japan. The UV-visible spectra are also investigated by UV-T80 series of UV-Visible spectrophotometer (PG instruments Limited, Leicestershire, UK). The magnetization/field correlation was evaluated using a vibrating sample magnetometer (VSM) (7400 Series VSM, Lake Shore Cryotronics, Inc., OH, USA) to determine the magnetic characteristics and hysteresis loops. The magnetic characteristics that were measured consisted of saturation magnetization, retentivity, and coercivity. Due to the dependence of magnetization saturation and retentivity on the weight and volume of the sample, the acquired values were normalized by dividing them by the sample’s weight. The magnetization was measured as a function of the applied field for all samples at room temperature. The magnetometer was calibrated using a standard nickel sphere. The analyses were conducted utilizing a maximum applied field of 2 Tesla.

Ferroportin Gene Expression Analysis

The quantitative polymerase chain reaction (qPCR) was used to determine the expression level of the gene encoding the iron-exporting protein Ferroportin (Solute carrier family 40 member 1, SLC40A1) in three leukemia cell lines: Kasumi-1 (representing acute myeloblastic leukemia), HL-60 (representing acute promyelocytic leukemia), and THP-1 (representing acute monocytic leukemia). The expression level was compared to that of normal hematopoietic cells (primary bone marrow CD34+ cells). Concisely, the cells were collected by rinsing with phosphate buffered saline (PBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), treated with 0.25% trypsin in PBS to detach them, and then spun at 300 x g for 5 minutes at 4°C. 1×107 cells were used to isolate total RNA using the RNeasy Plus Mini Kit (Cat. No. 74134, Qiagen, Hilden, Germany) following the instructions provided by the manufacturer. The reverse transcription process involved converting approximately 1 µg of RNA into complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (Cat. No. 205311, Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. The gene expression levels were quantitatively assessed using the QuantiTect SYBR Green PCR Kit (Cat. No. 204143, Qiagen, Hilden, Germany), in accordance with the manufacturer’s recommendations. The QuantiTect Primer Assay (Cat. No. 249900, Qiagen, Hilden, Germany) was also used. The qPCRs were conducted using the Applied Biosystems QuantStudio 3 Real-Time PCR System, manufactured by Thermo Fisher Scientific in Waltham, MA, USA. The comparative Ct method (2−ΔΔCt) was employed to examine relative gene expression, with ABL1 non-receptor tyrosine kinase serving as the endogenous reference gene. The gene expression levels were quantified in three replicates and shown as the ratio of gene expression in leukemia cells compared to the expression levels of the endogenous control, related to the normal primary bone marrow CD34+ cells. The results are reported as the mean ± standard deviation.

In vitro Anti-Proliferative Activity

The GA-stabilized/destabilized Fe3O4 NPs and CNTs were tested for their cytotoxic effects on normal primary bone marrow CD34+ cells (ATCC PCS-800-012) and three leukemia cell lines (Kasumi-1 [ATCC CRL-2724], HL-60 [ATCC CCL-240], and THP-1 [ATCC TIB-202]), which were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured according to the manufacturer’s recommendations in either Iscove’s Modified Dulbecco’s Medium (IMDM) or RPMI 1640 Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The culture medium was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained at a temperature of 37°C and in an atmosphere containing 5% CO2. Cell viability was assessed by employing the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation Kit I (Cat. No. 11465007001, Roche, Basel, Switzerland) in accordance with the instructions provided by the manufacturer. In summary, cells were placed in 96-well flat-bottom microtiter plates with a density of 5×103 cells/well in 100 μL of culture media. They were then incubated for 24 hours at a temperature of 37°C and an environment containing 5% CO2. After the incubation time, solutions containing GA-stabilized/destabilized Fe3O4 NPs and CNTs, either separately or together, were mixed with culture media to create concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL. These concentrations were then administered to the cells. In the negative control wells, an equal volume of growth media was supplied to the untreated cells. The cytotoxicity of doxorubicin, a standard anticancer medication sold by Sigma-Aldrich, Merck, Darmstadt, Germany, was assessed under identical conditions as a positive control. The cells were exposed to Fe3O4 NPs and CNTs that were either stabilized or destabilized by GA, either individually or in combinations, for a duration of 72 hours at a temperature of 37°C and an environment containing 5% CO2. Following the incubation period, 10 μL of the MTT reagent (5 mg/mL in phosphate buffered saline) was introduced to each well, resulting in a final concentration of 0.5 mg/mL. The microtiter plates were then incubated for 4 hours at a temperature of 37°C in an environment containing 5% CO2. Subsequently, 100 μL of the solubilization solution was added to each well. The microtiter plates were then left overnight in the incubator at a temperature of 37°C and an environment containing 5% CO2. This was done to dissolve the purple formazan crystals. The samples were analyzed for absorbance at a wavelength of 570 nm using the xMark Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). Three cell viability assays were conducted for each cell line, with each nanoformulation tested in triplicate at a specific concentration in each assay. Cell viability was determined using the formula: cell viability (percentage) = (absorbancetreated / absorbanceuntreated) × 100. The inhibitory concentration 50% (IC50) of each nanoformulation was determined by measuring its in vitro growth inhibition effect. IC50 is defined as the concentration (μg/mL) at which the treated cells show a 50% growth inhibition compared to the untreated control cells. The IC50 values were calculated as the mean ± standard deviation (SD) of three independent measurements. The IC50 value was calculated using the GraphPad Prism 9.0 software (GraphPad, Dotmatics, Boston, MA, USA) by nonlinear regression curve fitting, specifically by plotting Log [concentration] against normalized response.

Determination of Intracellular Iron Content

The impact of GA-stabilized/destabilized Fe3O4 NPs and CNTs, either individually or in combinations, on the levels of iron inside the cells was evaluated by subjecting the cells to the predetermined IC50 concentration of each nanoformulation for a duration of 48 hours at a temperature of 37°C and a CO2 concentration of 5%. To the untreated cells, an equal volume of PBS was introduced. The quantification of total iron content was performed in the cell lysates using a colorimetric technique. In summary, cells were collected by centrifugation at 500 x g for a duration of 5 minutes at a temperature of 4°C. Subsequently, the cells were rinsed twice with chilled PBS. Afterwards, the liquid portion above was discarded and the solid mass of cells was gathered. The cells were broken down using a Cell Extraction Buffer (containing Tris, NaCl, EDTA, EGTA, NaF, Na4P2O7, Na3VO4, Triton X-100, glycerol, SDS, and deoxycholate) provided by Invitrogen, Thermo Fisher Scientific. The buffer was supplemented with a Protease Inhibitor Cocktail from Sigma-Aldrich, Merck KGaA, and Phenylmethanesulfonyl fluoride (PMSF) from Sigma-Aldrich, Merck KGaA. The cells were kept on ice for 30 minutes at a concentration of 1×107 cells/mL, following the instructions provided by the manufacturer. The lysates were clarified using centrifugation at a speed of 13,000 rpm for a duration of 10 minutes at a temperature of 4°C. The Bio-Rad Protein Assay was used to assess the concentration of total protein in the cleared cell lysates, following the directions provided by the manufacturer. After the cell lysates were cleaned, they were divided into smaller portions and frozen at a temperature of −80°C for future study. The Iron Assay Kit (ab83366, Abcam, Cambridge, UK) was used to estimate the total iron levels, following the manufacturer’s instructions. The intracellular iron contents were quantified in triplicate, adjusted for sample dilution, standardized to the total protein content of the cell lysates, and reported as nmol/mg total protein of cell lysate (mean ± SD, n=3).

Measurement of Intracellular ROS Levels

The levels of reactive oxygen species (ROS) within the cells were measured using the Fluorometric Intracellular ROS Kit (Cat. No. MAK143, Sigma-Aldrich, Merck, Darmstadt, Germany) as per the instructions provided by the manufacturer. The test relies on the idea that ROS interact with a fluorogenic sensor located in the cytoplasm, leading to the production of a fluorometric product that is directly proportional to the quantity of ROS present. In summary, the cells were placed in 96-well microplates with poly-D-lysine coating, black walls, and optically clear flat bottoms. The cells were seeded at a density of 50×103 cells per well, with each well containing 90 μL of culture media. The cells were then incubated for 24 hours at a temperature of 37°C and in an environment with 5% CO2. Subsequently, 100 μL of the Master Reaction Mix was introduced into each well, and the cells were subjected to incubation for 1 hour at a temperature of 37°C in an environment containing 5% CO2. Subsequently, the cells were exposed to the predetermined IC50 concentration of each nanoformulation, which was dissolved in 20 μL of PBS per well. Equivalent volume of PBS was added to the negative control wells, which contained untreated cells. The ability of doxorubicin (Adriamycin; Sigma-Aldrich, Merck, Darmstadt, Germany) to produce ROS was assessed using the same experimental settings as a positive control. Following a further 48-hour incubation at a temperature of 37°C and a CO2 concentration of 5%, the fluorescence intensity was quantified using the FluoStar Omega multi-mode microplate reader (BMG Labtech, Ortenberg, Germany). The reader was set to an excitation wavelength of 490 nm and an emission wavelength of 525 nm (λex= 490/λem = 525 nm). The experiment was conducted in triplicates for each nanoformulation in every cell line. ROS levels were quantified by calculating the fold change of relative fluorescence units (RFU) in treated cells compared to the negative control (untreated cells). The results were reported as the mean ± standard deviation (SD) of three separate measurements.

Mechanistic Investigations

In order to investigate the molecular mechanisms responsible for the cytotoxic effects of the prepared nanostructures on leukemia cell lines, we treated the most sensitive cell line (HL-60) with the predetermined IC50 of each nanoformulation. We then conducted various tests to determine the mechanisms involved, including estimating the number of apoptotic cells, analyzing the distribution of cell-cycle phases, measuring the levels of apoptosis-related proteins, and quantifying the expression levels of genes encoding oxidative stress-related proteins and cyclin-dependent kinase inhibitors.

Determination of Apoptotic Cells

In order to determine if the prepared nanostructures have a toxic effect on leukemia cell lines by causing apoptosis, the HL-60 cell line, which is the most responsive, was exposed to each nanoformulation for 48 hours at a temperature of 37°C and a CO2 concentration of 5%. The percentage of cells undergoing apoptosis was measured using the BD FITC Annexin V Apoptosis Detection Kit I (Cat. No. 556547, BD Biosciences, San Jose, CA, USA) following the instructions provided by the manufacturer. In summary, the cells were rinsed twice with chilled PBS and thereafter subjected to centrifugation at 300 x g for 5 minutes at ambient temperature. The liquid portion was removed and the clumped cells were mixed again in a solution called 1X Annexin V Binding Buffer, with a concentration of 1×106 cells per milliliter. 100 µL of the cell solution was transferred to a 5 mL tube, and then 5 µL of FITC Annexin V and 5 µL of propidium iodide (PI) were added. The cells were gently agitated and placed in a dark environment at room temperature for a duration of 15 minutes. Afterwards, 400 µL of 1X Annexin V Binding Buffer was introduced, and the cells were collected using a BD Accuri C6 Flow Cytometer and examined using BD Accuri C6 software (BD Biosciences, San Jose, CA, USA).

Estimation of Cell-Cycle Phase Distribution

In order to assess the impact of the prepared nanostructures on the distribution of cell-cycle phases, the HL-60 cell line, which exhibited the highest sensitivity, was exposed to each nanoformulation for a duration of 48 hours at a temperature of 37°C and a CO2 concentration of 5%. The distribution of cell-cycle phases was then determined using the BD CycleTest Plus DNA Reagent Kit (Cat. No. 340242, BD Biosciences, San Jose, CA, USA) following the instructions provided by the manufacturer. Concisely, the cells were rinsed twice with Buffer Solution, and thereafter subjected to centrifugation at 300 x g for 5 minutes at room temperature. The liquid portion was discarded, and the solid portion containing cells was mixed with Buffer Solution to achieve a concentration of 1×106 cells per milliliter. The cell solution was thereafter subjected to centrifugation at 400 x g for a duration of 5 minutes at ambient temperature, following which the liquid portion above the sediment was discarded. The lipids of the cell membrane were dissolved using a nonionic detergent. The cell cytoskeleton and nuclear proteins were removed using trypsin. Cellular RNAs were broken down using RNase. The nuclear chromatin was stabilized using spermine. Finally, the DNA was stained with propidium iodide (PI) according to the instructions provided by the manufacturer. The cells were collected using a BD Accuri C6 Flow Cytometer and examined using BD Accuri C6 software (BD Biosciences, San Jose, CA, USA).

Determination of Apoptosis-Related Proteins Level

The impact of the produced nanostructures on apoptosis-related proteins was evaluated by subjecting the most susceptible cell line (HL-60) to each nanoformulation for 48 hours at a temperature of 37°C and a CO2 concentration of 5%. The levels of apoptosis-associated proteins, such as p53, Bax, Bcl-2, Cytochrome C, and Caspase-3, were measured in the cell lysates using Enzyme-Linked Immunosorbent Assays (ELISAs). In summary, cells were collected by centrifugation at 500 x g for a duration of 5 minutes at a temperature of 4°C. The cells were subsequently rinsed twice with chilled PBS. Afterwards, the liquid portion above the sediment was discarded and the solid mass of cells was gathered. The cells were lysed in a Cell Extraction Buffer containing various components including Tris, NaCl, EDTA, EGTA, NaF, Na4P2O7, Na3VO4, Triton X-100, glycerol, SDS, and deoxycholate. The lysing process was carried out on ice for 30 minutes at a concentration of 1×107 cells per milliliter of Cell Extraction Buffer. A Protease Inhibitor Cocktail and Phenylmethanesulfonyl fluoride (PMSF) were added to the buffer as instructed by the manufacturer. The Cell Extraction Buffer was obtained from Invitrogen, Thermo Fisher Scientific, and the Protease Inhibitor Cocktail and PMSF were obtained from Sigma-Aldrich, Merck KGaA. The lysates were clarified using centrifugation at a speed of 13,000 rpm for a duration of 10 minutes at a temperature of 4°C. The Bio-Rad Protein Assay was used to assess the concentration of total protein in the cleared cell lysates, following the directions provided by the manufacturer. The cell lysates were cleaned, divided into smaller portions, and frozen at a temperature of −80°C for future ELISAs. We used commercially available ELISA kits to measure the concentrations of apoptosis-related proteins. These kits included the Human p53 ELISA Kit (ab171571, Abcam, Cambridge, UK), Human Bax ELISA Kit (ab199080, Abcam, Cambridge, UK), Human Bcl-2 ELISA Kit (ab272102, Abcam, Cambridge, UK), Human Cytochrome C ELISA Kit (Cat. No. BMS263, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and Human cleaved (active) Caspase-3 ELISA Kit (Cat. No. KHO1091, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). We followed the protocols provided by the manufacturers. The levels of apoptosis-related proteins were quantified in three replicates, adjusted for sample dilution, standardized to the overall protein content of the cell lysates, and reported as ng/mg total protein of cell lysate (mean ± SD, n=3).

Gene Expression Quantitation

The impact of the prepared nanostructures on the expression of genes that encode proteins related to oxidative stress (SIRT1, FOXO3, NFE2L2, and MAP3K5) and genes that encode inhibitors of cyclin-dependent kinases (p21 [CDKN1A] and p27 [CDKN1B]) was investigated. This was done by subjecting the most susceptible cell line (HL-60) to each nanoformulation for 48 hours at a temperature of 37°C and a CO2 concentration of 5%. Quantitative polymerase chain reaction (qPCR) was used to evaluate the gene expression levels of SIRT1, FOXO3, NFE2L2, MAP3K5, CDKN1A, and CDKN1B. Concisely, the cells were collected by rinsing with PBS, treated with 0.25% trypsin in PBS to detach them, and then subjected to centrifugation at 300 x g for 5 minutes at 4°C. 1×107 cells were used to extract total RNA using the RNeasy Plus Mini Kit (Cat. No. 74134, Qiagen, Hilden, Germany) following the instructions provided by the manufacturer. The reverse transcription process involved converting approximately 1 µg of RNA into complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (Cat. No. 205311, Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. The gene expression levels were quantitatively assessed using the QuantiTect SYBR Green PCR Kit (Cat. No. 204143, Qiagen, Hilden, Germany), in accordance with the manufacturer’s recommendations. QuantiTect Primer Assays (Cat. No. 249900, Qiagen, Hilden, Germany) were also used. The qPCRs were conducted using the Applied Biosystems QuantStudio 3 Real-Time PCR System, manufactured by Thermo Fisher Scientific in Waltham, MA, USA. The analysis of relative gene expression was conducted using the comparative Ct method (2−ΔΔCt), with ABL1 non-receptor tyrosine kinase serving as the endogenous reference gene. The gene expression levels were quantified in three replicates and reported as the ratio of gene expression in leukemia cells compared to the expression levels of the endogenous control, relative to the untreated cells. The results are presented as the mean ± standard deviation.

Statistical Analysis

The normality of the data distribution was assessed using the Kolmogorov–Smirnov test. The results were presented as the mean ± standard deviation (SD) and were compared using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. The statistical analyses of the data were performed using the GraphPad Prism 9.0 program (GraphPad, Dotmatics, Boston, MA, USA).

Results

Herein, an effective and direct approach was described for the simple combination of high-content superparamagnetic Fe3O4 nanoparticles of definite size uniformly deposited on the surface of high-mechanically strength multi-walled CNTs as nanocontainer template which is dispersed and stabilized in aqueous solution of biocompatible Gum Arabic biopolymer. Our results suggest a novel and simple approach for controlling the loading and size distribution of Fe3O4 on the surface of multi-walled CNTs, which leads to further development of CNT-based nanomaterials. Noteworthy, although numerous studies have been conducted to examine the biological effects of magnetite (Fe3O4) NPs and CNTs, the cytotoxic effects of Fe3O4 NPs and CNTs remain inconsistent. The current state of knowledge is inconclusive, as there are a multitude of contrasting results. Although several studies have suggested that cytotoxicity is minimal or nonexistent, others have reported significant cytotoxic effects. Additionally, the potential deleterious effects and the underlying molecular pathways of Fe3O4 NPs and CNTs on AML cells are still not fully understood.

Surface Treatments of CNTs

The initial stage of the treatment procedure for the as received CNTs was subjecting them to a temperature of 450°C in an oven for a duration of 30 minutes. This was done to eliminate certain volatile organic substances, such as the binding agent, that are introduced into the particles during the manufacturing process. Following the heat treatment of the CNTs, they were subjected to a washing process using acetone to eliminate any soluble organic contaminants that may have been generated during the heat treatment. The CNTs underwent acid treatments using a combination of H2SO4 and HNO3. The TEM image in Figure 2a displays the multi-walled CNTs under investigation, with an approximate hollow core of inner diameter of 4.5 nm and outer diameter of 19 nm with a multiwall graphitic structure. Figure 2b displays the X-ray diffraction (XRD) pattern of the examined CNTs. The diffraction peaks were observed at angles of 26°, 43°, 53°, and 79°, which correspond to the crystalline planes 002, 100, 004, and 110, respectively. These assignments are based on the joint committee on powder diffraction standards (JCPDS) # 96–101-1061.

Figure 2 TEM (a) and XRD pattern (b) of the investigated CNTs.

Synthesis and Stabilization of Fe3O4 and Fe3O4/CNTs Nano-Hybrid Composite in GA

The process of depositing Fe3O4 NPs onto the surface of CNTs was investigated in the presence of ultrasonic radiation. The deposition solution consisted of essential components, namely ferrous sulphate heptahydrate and ferric chloride hexahydrate, which included salts of ferrous and ferric ions. Ammonium hydroxide was employed as a precipitating agent. For the precipitation process using ammonia solution, it is necessary to adjust the pH of the solution to approximately 12.5 and maintain a temperature of around 67°C. Figure 3 displays the produced Fe3O4 NPs, along with the Fe3O4/CNTs nanocomposite, which is evenly distributed in the GA aqueous solution. The results suggest that 1 wt. % GA aqueous solution is a suitable stabilizing agent for the synthesized NPs. The Fe3O4 and the Fe3O4/CNTs NPs were evenly distributed over an extended duration.

Figure 3 The produced Fe3O4 NPs and the Fe3O4/CNTs nano-hybrid composite dispersed and stabilized in GA solution.

FTIR and UV-Visible Spectrum of the Nano-Hybrid Composite

GA was employed as a stabilizing agent for the synthesized Fe3O4 and Fe3O4/CNTs nanocomposite particles, facilitating their dispersion in aqueous solutions and resulting in the formation of a stable colloidal solution. Figure 4a and b displays the FTIR spectrum illustrating the various functional groups found in the GA as well as after the combinations with the prepared nanoparticles (Fe3O4 and Fe3O4/CNTs). Also Figure 4c represent the absorption peaks of the UV-Visible spectra for the investigated nanoparticles stabilized with GA in aqueous solution. Figure 5 depicts a schematic illustration of the anticipated interaction between the carbohydrate and glycoprotein chains of the GA and the Fe3O4/CNTs nanocomposite particles.

Figure 4 FTIR spectrum of the investigated GA (a) and the prepared nanoparticles stabilized by GA (b), and the UV-visible spectrum (c) of all the investigated nanoparticles.

Figure 5 Schematic diagram of the interaction between the carbohydrate and the glycoprotein chains of the GA stabilizing agent and the synthesized Fe3O4 nanocomposite particles.

Particle Shape, Size and Morphology of the NPs

SEM micrographs of the deposited Fe3O4 NPs at various magnifications and particles size distribution are presented in Figure 6a and b. The Fe3O4 NPs that were deposited have a nearly spherical morphology, with a particle size ranging from 28 to 49 nm. Furthermore, the nanoscale Fe3O4 NPs were formed and made stable in the aqueous solution containing the GA stabilizing agent. The optimal reaction temperature of around 67°C was determined by monitoring the course of the reaction and ensuring that all iron ions in the solution were transformed into Fe3O4 NPs. Figure 7 displays high-resolution scanning electron microscopy micrographs of the manufactured Fe3O4/CNTs nanocomposite particles, together with both low and high magnifications. Additionally, the figure includes EDS compositional analysis of the particles. The findings revealed that the acid functionalized CNTs were adorned with the Fe3O4 NPs. The EDS compositional analysis indicated that the produced Fe3O4/CNTs nanocomposite particles composed primarily of iron metal and elemental oxygen, with a composition of 1 wt.% Fe3O4/CNTs.

Figure 6 SEM of the prepared Fe3O4 NPs where (a) high magnification image and (b) high magnification image of particles size distribution.

Figure 7 SEM of GA-stabilized Fe3O4 NP with low and high magnifications (a and b), and EDS compositional analysis of GA-stabilized Fe3O4 NPs (c).

XRD and Phase Analysis of the NPs

Figure 8 displays the XRD patterns of both the Fe3O4 NPs and the Fe3O4/CNTs nanocomposites. The results indicate that the observed peaks are caused by the existence of specific diffraction peaks, namely 111, 220, 311, 222, 400, 422, 511, 440, 531, 620, 533, 622, and 444. These peaks correspond to the JCPDS 79–0419 of the Fe3O4 structure. No extra or foreign peaks were detected. The results suggest that the prepared Fe3O4 NPs have a high level of purity. Furthermore, Figure 8 depicts the XRD pattern of the Fe3O4/CNTs nanocomposites. The results indicate that the detected peaks are a result of the presence of Fe3O4 NPs that were deposited onto the surface of the CNTs.

Figure 8 XRD patterns of the synthesized Fe3O4 and the Fe3O4/CNTs nanocomposite particles.

Magnetic Properties of the Nano-Hybrid Composite

Figure 9a–c depicts the hysteresis loops of the examined CNTs, Fe3O4 NPs, and the nanocomposite particles of Fe3O4/CNTs. The saturation magnetizations (Ms), retentivity (Mr), and coercivity (Hc) of the studied CNTs, Fe3O4 NPs, and Fe3O4/CNTs nanocomposite were determined at room temperature using a maximum applied field of 2T, as presented in Figure 9. The Ms value of Fe3O4/CNTs nanocomposite particles (52.172 emu/g; see Figure 9c) is lower than that of the Fe3O4 NPs (57.741 emu/g; see Figure 9b), possibly because of the influence of the 1 wt. % content of CNTs (Ms 2.0269 emu/g; see Figure 9a) in the Fe3O4/CNTs nanocomposite particles. Additionally, the Fe3O4 sample at the nanoscale exhibits superparamagnetism and a saturation magnetization (Ms) value that is lower than that of the bulk Fe3O4 sample (92 emu/g). In addition, Figure 9 illustrates the hysteresis vanished with a little retentivity value of 0.17078 emu/g and a coercivity (Hc) of 117.99 G, 1.40040 emu/g and coercivity (Hc) of 38.913 G, 0.80027 emu/g and coercivity (Hc) of 27.054 G in case of CNTs (see Figure 9a), Fe3O4 NPs (see Figure 9b) and Fe3O4/CNTs nanocomposite particles (see Figure 9c), respectively. This suggests that there is no long range of magnetic dipole-dipole interaction among the assemblies of the superparamagnetic NPs. Consequently, the Fe3O4 NPs and Fe3O4/CNTs nanocomposite particles exhibit magnetization easily when exposed to an applied magnetic field, with retentivity values of 1.40040 emu/g (see Figure 9b) and 0.80027 emu/g (see Figure 9c), respectively. A potential cause for this distinct structure is the independent thermal fluctuation of minuscule ferromagnetic domains within the particles.

Figure 9 Magnetic hysteresis loops of the investigated CNTs (a), as well as the prepared Fe3O4 NPs (b) and the Fe3O4/CNTs nano-hybrid composite (c).

SLC40A1 Expression Analysis

The gene SLC40A1, which codes for the protein ferroportin that exports iron, was found to have significantly lower levels of expression in the myeloid leukemia cell lines studied (Kasumi-1, HL-60, and THP-1) compared to normal primary bone marrow CD34+ cells. Among the cell lines, HL-60 had the lowest expression, followed by THP-1 and Kasumi-1 (Figure 10).

Figure 10 SLC40A1 gene expression levels in leukemia cell lines. Expression levels were measured in triplicate, and expressed as the fold change in gene expression in the treated cells normalized to the expression levels of the endogenous control and relative to normal primary bone marrow CD34+ cells. Data are represented as mean ± SD of three independent experiments. SLC40A1 gene expression in Kasumi-1, HL-60 and THP-1 was significantly (p < 0.05) lower than that in primary bone marrow CD34+ cells.

In vitro Cytotoxic Activity

A 72-hour treatment of normal hematopoietic cells (primary bone marrow CD34+) or leukemia cell lines (Kasumi-1, HL-60, or THP-1) with varying concentrations of each nanoformulation revealed that GA alone did not have a significant impact on cell viability. This suggests that GA itself is nearly non-cytotoxic to both leukemia cell lines and normal hematopoietic cells. The combination of GA-stabilized/destabilized Fe3O4 NPs and CNTs, as well as their individual forms, exhibited notable cytotoxicity against the leukemia cell lines under investigation. The IC50 values for these formulations ranged from 42.437 to 189.842 μg/mL. Remarkably, GA-Fe3O4 NPs/CNTs exhibited the highest level of cytotoxicity, as evidenced by IC50 values of 42.437, 63.343, and 141.674 μg/mL in HL-60, THP-1, and Kasumi-1 cell lines, respectively. HL-60 cells had the highest sensitivity to growth inhibition caused by the investigated nanostructures, with an IC50 range of 42.437‒96.901 μg/mL. THP-1 cells followed with an IC50 range of 57.681‒138.465 μg/mL, and Kasumi-1 cells had an IC50 range of 90.725‒189.842 μg/mL. Although the evaluated nanostructures showed significant growth inhibitory effect against the leukemia cell lines (IC50: 42.437‒189.842 μg/mL), they exhibited comparatively minimal cytotoxicity against normal hematopoietic cells (IC50: 113.529‒162.656 μg/mL). Compared to Fe3O4 NPs or CNTs used separately, the combination of Fe3O4 NPs and CNTs showed stronger growth inhibitory action. The IC50 values for this combination were 51.882 μg/mL in HL-60 and 57.681 μg/mL in THP-1. In contrast, Fe3O4 NPs alone exhibited more efficacy in reducing the growth of Kasumi-1 cells compared to either Fe3O4 NPs combined with CNTs or CNTs alone. It is worth mentioning that the combination of Fe3O4 NPs with CNTs in a nanocomposite significantly improved their ability to fight against leukemia cells, with the effectiveness varying depending on the kind of cell. Furthermore, the use of GA to stabilize Fe3O4 NPs, CNTs, and Fe3O4 NPs/CNTs can enhance their ability to inhibit cellular proliferation, with the extent of this effect varying depending on the type of cell. Detailed anti-proliferative activity of the prepared nanostructures, against normal hematopoietic and leukemia cell lines is shown in Table 1.

Table 1 In vitro Cytotoxic Activity of GA, Fe3O4, CNTs, Either Alone or in Combinations, Against Leukemia Cell Lines and Normal Hematopoietic Cells

Intracellular Iron Levels

A considerable increase in the amount of iron within the cells of the myeloid leukemia cell lines was seen after treating them with nanostructures containing Fe3O4 for 48 hours. Among the cell lines tested, Kasumi-1 exhibited the greatest levels of iron, followed by THP-1 and HL-60 (Figure 11A).

Figure 11 Effect of GA-stabilized/destabilized Fe3O4 NPs and CNTs, either alone or in combinations, on intracellular iron content (A) and ROS level (B) in leukemia cell lines. In all cell lines treated with Fe3O4-containing nanostructures, iron content was significantly (p < 0.05) higher than that in untreated cells. ROS level in all cell lines treated with GA-stabilized/destabilized Fe3O4 NPs and CNTs, either alone or in combinations, was significantly (p < 0.05) higher than that in untreated cells.

Intracellular ROS Concentrations

Quantifying intracellular ROS levels 48 hours after treatment with nanostructures revealed that HL-60 cells were the most influenced, with the highest elevation in ROS levels. This observation is consistent with the lowest expression of SLC40A1, as depicted in Figure 11B.

Mechanistic Investigations Effect of the Prepared Nanoformulations on Apoptosis Induction

Fe3O4, CNTs, Fe3O4/CNTs, GA-Fe3O4, GA-CNTs, and GA-Fe3O4/CNTs all showed a large rise in early apoptotic HL-60 cells. The percentage of early apoptotic cells went from 0.04% in PBS-treated cells to 41.83%, 27.56%, 50.13%, 44.29%, 32.64%, and 54.58%, respectively. Additionally, doxorubicin (DXR) also resulted in a remarkable increase in early apoptotic cells, with a percentage of 47.92%. Similarly, the proportion of cells experiencing late apoptosis significantly increased from 0.13% in cells treated with PBS to 10.10%, 4.58%, 12.90%, 10.69%, 11.83%, and 11.85%, respectively. In contrast, DXR considerably boosted the percentage of late apoptotic cells to 9.50%. The results indicate that the produced nanostructures have a cytotoxic effect, partially through the activation of apoptosis (Figure 12A).

Figure 12 Effect of GA, Fe3O4, CNTs, either alone or in combinations, on apoptosis induction (A) and cell cycle progression (B) in in HL-60 cells. Data are represented as average percentage of three independent experiments.

Effect of the Prepared Nanoformulations on Cell Cycle Progression

Fe3O4, CNTs, Fe3O4/CNTs, GA-Fe3O4, GA-CNTs, and GA-Fe3O4/CNTs significantly increased the proportion of HL-60 cells in the G0/G1 phase from 53.4% in cells treated with PBS to 65.7%, 72.3%, 81.2%, 62.5%, 70.7%, and 79.0%, respectively. The population of cells in the S phase decreased significantly from 30.9% in PBS-treated cells to 22.2%, 18.2%, 11.9%, 25.0%, 18.5%, and 13.8%, respectively. Similarly, the population of cells in the G2/M phase decreased from 13.5% in PBS-treated cells to 10.0%, 7.5%, 4.5%, 11.2%, 8.3%, and 5.6%, respectively. The results suggest that the produced nanostructures may inhibit cell proliferation by causing cell cycle arrest at the G1 phase. Conversely, DXR significantly augmented the number of cells in the G2/M phase to 47.4%, while simultaneously reducing the population of cells in the S phase to 18.2% (Figure 12B).

Influence of the Prepared Nanoformulations on Apoptosis-Related Proteins

Treating HL-60 cells with different nanostructures, such as Fe3O4, CNTs, Fe3O4/CNTs, GA-Fe3O4, GA-CNTs, or GA-Fe3O4/CNTs, resulted in a significant increase in the level of the tumor suppressor protein p53. The increase was 4.67-fold, 0.86-fold, 2.54-fold, 3.80-fold, 0.22-fold, and 1.85-fold, respectively, compared to cells treated with PBS. Additionally, treatment with DXR caused a 4.53-fold increase in p53 concentration. The nanostructures that were examined resulted in a significant rise in the concentration of the pro-apoptotic protein BAX. The increases in BAX level were 0.63-fold, 3.44-fold, 2.02-fold, 1.24-fold, 5.16-fold, and 2.68-fold, respectively, compared to cells treated with PBS. In comparison, DXR caused a 3.01-fold increase in BAX levels. Similarly, the nanostructures that were tested resulted in a significant increase in the amount of the electron transport chain protein cytochrome C in the. The increase was 5.95-fold, 3.49-fold, 11.10-fold, 4.30-fold, 2.70-fold, and 7.02-fold, respectively, compared to cells treated with PBS. In contrast, DXR caused a 2.67-fold increase in the concentration of cytochrome C. In the same way, the nanostructures that were tested caused a significant increase in the concentration of caspase-3, an executioner protein of apoptosis, by 90082.33-, 56,415.67-, 135,465.67-, 110,799.00-, 72,115.67- and 163815.67-fold compared to cells treated with PBS. Additionally, DXR resulted in a 99715.67-fold increase in caspase-3 concentration. In contrast, the examined nanostructures did not have a notable suppressive impact on the levels of expression of the anti-apoptotic protein BCL-2 (Figure 13).

Figure 13 Influence of GA, Fe3O4, CNTs, either alone or in combinations, on apoptosis-related proteins, including p53 (A), BAX (B), BCL-2 (C), cytochrome C (D) and caspase 3 (E) in HL-60 cells. The concentrations of apoptosis-related proteins were measured in triplicate, corrected for sample dilution, normalized to the total protein content of the cell lysates, and expressed as ng/mg total protein of cell lysate. Data are represented as mean ± SD of three independent experiments. Compared to untreated cells, protein expression level increases in p53, BAX, cytochrome C and caspase 3 induced by treatment of the cells with GA-stabilized/destabilized Fe3O4 NPs and CNTs, either alone or in combinations, were statistically significant (p < 0.05).

Impact of the Prepared Nanoformulations on the Expression of the Genes Encoding Oxidative Stress-Related Proteins and Cyclin-Dependent Kinase Inhibitors

Exposing HL-60 cells to Fe3O4, CNTs, Fe3O4/CNTs, GA-Fe3O4, GA-CNTs, or GA-Fe3O4/CNTs, resulted in a notable increase in the expression of SIRT1, FOXO3, NFE2L2, MAP3K5, CDKN1A, and CDKN1B genes. This overexpression was more pronounced compared to the effect caused by DXR (Figure 14).

Figure 14 Impact of GA-stabilized/destabilized Fe3O4 NPs and CNTs, either alone or in combinations, on the expression of the SIRT1 (A), FOXO3 (B), NFE2L2 (C), MAP3K5 (D), CDKN1A (E) and CDKN1B (F) genes in HL-60 cells. Expression levels were measured in triplicate, and expressed as the fold change in gene expression in the treated cells normalized to the expression levels of the endogenous control and relative to the untreated cells. Data are represented as mean ± SD of three independent experiments. Compared to untreated cells, gene expression elevations in SIRT1, FOXO3, NFE2L2, MAP3K5, CDKN1A and CDKN1B induced by treatment of the cells with GA-stabilized/destabilized Fe3O4 NPs and CNTs, either alone or in combinations, were statistically significant (p < 0.05).

Discussion

The prepared Fe3O4/CNTs and Fe3O4/CNTs-GA nano-hybrid composite can be used as nanocontainers for different human medical treatments, including magnetic hyperthermia, and as drug delivery/carrier/release in the treatment of tumors. Further, Gum Arabic, a natural polymer known for its usage in controlled drug deliver