Rombus-shaped α-Fe₂O₃ nanoparticles for antibacterial and anticancer applications

ABSTRACT

Background

The increasing incidence of bacterial infections in cancer patients, combined with the growing limitations of conventional antibiotics such as poor site-specific targeting and antibiotic resistance, necessitates the development of advanced therapeutic strategies.

Methodology

Rhombus-shaped α-Fe₂O₃ nanoparticles (NPs) were synthesized via hydrothermal route and characterized for their structural, optical, and morphological properties. Lymecycline was encapsulated into NPs, and its pH-dependent release was assessed. Antibacterial activity was evaluated using the well diffusion and minimum inhibitory concentration assay, while anticancer potential was examined using AlamarBlue and cytotoxicity assays against THP-1 cells. Biocompatibility was assessed using normal L-929 fibroblast cells.

Results

The synthesized Fe₂O₃ NPs measured ~80 to 150 nm in length and ~50 nm in width. Lymecycline-loaded NPs demonstrated pH-responsive release, with 60% drug release at pH 5.5 and 43% at pH 7.4. They exhibited enhanced cytotoxicity (72%) against THP-1 cancer cells, while showing good biocompatibility with L-929 normal cells. Additionally, strong antibacterial activity was observed against Escherichia coli and Staphylococcus aureus.

Conclusions

Lymecycline-loaded α-Fe₂O₃ NPs exhibited pH-responsive drug release, selective cytotoxicity toward THP-1 cancer cells, strong antibacterial efficacy, and good biocompatibility with normal cells. These findings highlight their dual functionality and potential as a promising nanoplatform for future anticancer and antimicrobial therapies.

GRAPHICAL ABSTRACT

Graphical abstract illustrates the loading of lymecycline in rhombus-shaped α-Fe₂O₃ NPs. The lymecycline loaded α-Fe₂O₃ NPs exhibit targeted antibacterial and anticancer activity, enhancing cytotoxicity in cancer cells while showing significant antibacterial efficacy.

1. Introduction

Bacterial infections are the second leading cause of mortality among cancer patients, with incidence rates increasing in recent years [1,2]. These infections are traditionally managed using broad-spectrum antibiotics such as tetracycline, ciprofloxacin, norfloxacin, methicillin, and azithromycin [3–5]. Recently, there has been growing interest in repurposing antibiotics, particularly those targeting mitochondria such as tetracyclines for cancer therapy. These agents exhibit anti-inflammatory properties by inhibiting enzymes implicated in tumor progression, including matrix metalloproteinases [6], nitric oxide synthase [7], and neutrophil elastase [8,9]. Tetracycline, a well-known drug for broad-spectrum antibacterial activity, has also demonstrated cytotoxic effects through apoptosis induction in various cancer cell lines such as lymphocytes, osteosarcomas, and prostate cancer cells, thereby potentially limiting metastasis [10,11]. Lymecycline, a semi-synthetic derivative of tetracycline, is primarily used for its antibacterial and anti-acne effects. As it possesses a significantly enhanced solubility of ~5,000 times greater than the tetracycline base, it exhibits the highest oral bioavailability when compared to other tetracyclines [12,13]. However, its potential applications in cancer therapy remain largely unexplored. Furthermore, conventional antibiotics face several limitations such as non-specificity, systemic toxicity, misuse in non-bacterial infections, and most critically, the rapid emergence of antibiotic resistance.

Nanotechnology-based strategies have emerged as powerful tools in cancer resistance and bacterial infections. The development of different metal and metal oxide nanoparticles (NPs) such as gold, silver, zinc oxide (ZnO), and copper oxide (CuO) has markedly improved antimicrobial efficacy, especially in combating multidrug-resistant (MDR) microbial strains [14–16]. They exert their antimicrobial effects through multiple mechanisms, including the generation of reactive oxygen species (ROS), disruption of bacterial membrane integrity, and inhibition of essential cellular processes such as respiration and metabolic enzyme function [17,18]. Iron oxide nanoparticles (IONPs) have attracted significant research interest owing to their tunable magnetic and optical properties, chemical inertness, good biocompatibility, and multifunctionality [19]. Different polymorphic forms of IONPs such as magnetite (Fe₃O₄) [20], maghemite (γ-Fe₂O₃) [21], and hematite (Fe₂O₃) [22] have been extensively used for antimicrobial and anticancer activities [23]. IONPs exhibit broad-spectrum antimicrobial activity, demonstrating pronounced efficacy against both Gram-positive and Gram-negative bacterial strains, as well as a range of fungal species. Notably, their intrinsic versatility allows IONPs to be functionalized through surface coating or encapsulation with antibiotics, thus enabling precise drug delivery, targeted therapy, and improved pharmacokinetics. Additionally, they serve as effective agents in cellular imaging, cell tracking, and labeling applications due to their magnetic properties and biocompatibility [24].

To date, many physical and chemical synthesis techniques have been employed to produce iron oxide NPs ranging from co-precipitation [25] and mechanochemical methods [26] to wire electrical explosion [27] and electrochemical approaches [28]. However, many of these methods suffer from significant drawbacks such as the use of toxic reagents, hazardous reaction conditions, and the requirement for high energy input and/or vacuum systems. These limitations highlight the urgent need for environmentally benign and sustainable synthesis strategies.

Encapsulation of antibiotics within NPs significantly enhances their therapeutic efficacy by improving pharmacokinetic profiles, prolonging systemic circulation time, enabling controlled and sustained drug release, and reducing systemic exposure and dosing frequency. This targeted delivery approach facilitates the preferential accumulation of antibiotics at infection sites or tumor microenvironments, thereby maximizing therapeutic outcomes while minimizing off-target effects and associated toxicity [29]. Furthermore, the engineered NPs have demonstrated the capacity to overcome physiological and biological barriers ranging from biofilm matrices to intracellular microbial reservoirs that commonly limit the effectiveness of conventional therapies [13,29,30]. Especially for cancer therapy, NPs such as poly(lactic-co-glycolic acid) [31,32], chitosan [33], and solid lipids [34] have been demonstrated for targeted delivery to tumor cells, thereby minimizing collateral damage to healthy tissue. Additionally, tetracycline-loaded in different platforms such as graphene oxide [35], nickel ferrite [13], mesoporous silica [36], and calcium phosphate [37] have shown significant antibacterial and anticancer activity by improving drug stability, bioavailability, and efficacy against MDR strains.

In this study, we report on the development of a lymecycline-loaded nanocarrier system based on rhombus-shaped α-Fe₂O₃ (hematite) NPs for dual anticancer and antibacterial applications. A facile hydrothermal synthesis method was employed to fabricate the rhombus-shaped α-Fe₂O₃ NPs, utilizing ferric chloride and sodium molybdate as precursor materials. The successful encapsulation of lymecycline within the α-Fe₂O₃ nanostructures and its subsequent controlled release were thoroughly investigated. The therapeutic potential of the resulting formulation was systematically evaluated through in vitro assays, demonstrating promising efficacy in both antimicrobial and anticancer therapy. The anticancer activity and biocompatibility of the lymecycline-loaded α-Fe₂O₃ NPs were assessed in vitro using human monocytic leukemia (THP-1) and L-929 fibroblast cell lines. Further, the antibacterial activity was investigated against both Gram-positive and Gram-negative pathogenic bacterial strains using agar-well diffusion and minimum inhibitory concentration (MIC) assays. Thus, this work aims to enhance the therapeutic potential of α-Fe₂O₃-based nanocarriers by leveraging their drug delivery capability in combination with the broad-spectrum activity of lymecycline. Notably, to the best of our knowledge, this is the first report on the cytotoxic and antimicrobial evaluation of lymecycline-encapsulated rhombus-shaped α-Fe₂O₃ NPs, thereby demonstrating their potential as multifunctional therapeutic agents.

2. Materials and methods

2.1. Chemicals

Ferric chloride (FeCl₃) anhydrous (purity ≥98%), sodium hydroxide (NaOH), sodium acetate anhydrous (C₂H₃NaO₂), acetic acid (CH₃COOH), and hydrochloric acid (HCl) were obtained from the Sisco Research Laboratory (SRL), India. Sodium molybdate dihydrate (Na₂MoO₄·2 H₂O) and diphenyl-1-pyrenylphosphine (DPPP) were procured from Sigma-Aldrich, India. Mueller–Hinton Agar (MHA), Mueller–Hinton Broth (MHB), Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI 1640) medium, acridine orange (AO), and propidium iodide (PI), and Alamar Blue were all purchased from HIMedia Laboratories, India. Fetal bovine serum (FBS, Gibico), 1% penicillin, 1% gentamicin, dimethyl sulfoxide (DMSO), and phosphate-buffered saline (PBS), glutathione (GSH), nicotinamide adenine dinucleotide 2´-phosphate reduced tetrasodium salt hydrate (NADPH), and sodium azide were procured from Sisco Research Laboratory, India. All experiments were performed using Milli-Q water with a resistivity ≥18 MΩ cm.

2.2. Synthesis of rhombus-shaped α-Fe₂O₃ NPs

Rhombus-shaped α-Fe₂O₃ NPs were synthesized using a hydrothermal method. 30 mg of ferric chloride was dissolved in 50 mL of milli-Q water, followed by the addition of 1.8 mM of sodium molybdate solution (1 mL). After mixing these solutions under continuous stirring at 1200 rpm for 1 h under ambient conditions, the pH of the solution was adjusted to 9 using 0.1 M NaOH. Later, the solution was transferred to a Teflon-lined stainless-steel autoclave and heated to 180°C for 12 h in a hot air oven to synthesize rhombus-shaped α-Fe₂O₃ NPs. After the reaction, the autoclave was allowed to cool naturally to room temperature. Upon cooling, the reddish-brown precipitate formed was collected by centrifugation at 10,000 rpm for 5 min and washed thoroughly three times with milli-Q water and ethanol. The final product of Fe₂O₃ NPs was then dried overnight in a hot-air oven at 60°C and stored for further analysis.

2.3. Characterization techniques

The rhombus-shaped α-Fe₂O₃ NPs were characterized using several analytical techniques to determine their optical, morphological, structural, and chemical properties. The absorbance of the α-Fe₂O₃ NPs was measured using a UV-visible spectrophotometer to evaluate the optical properties of the formed NPs (Nanodrop 2000c, Thermo Fisher Scientific, USA). The size and shape of the NPs were examined using high-resolution scanning electron microscopy operated at an accelerating voltage of 20 kV (HR-SEM, AperoS, Thermo Fisher Scientific, Singapore), and high-resolution transmission electron microscopy (HR-TEM, 2100 Plus, JEOL, Japan). The HR-TEM was operated at an accelerating voltage of 200 kV to accurately determine the nanoparticle morphology and acquire energy dispersive X-ray spectroscopy (EDS) spectrum. Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural information and functional groups present in the synthesized Fe₂O₃ NPs. The analysis was performed using a Nicolet 6700 FTIR spectrometer over the wavenumber range of 500–4000 cm⁻¹ in attenuated total reflection mode (Thermo Fisher Scientific, USA). The phase formation and crystallinity of the NPs was investigated using a powder X-ray diffraction technique (XRD, D/MAX-2550 PC X-ray diffractometer). The XRD patterns were recorded in the 2θ range of 10–80° to identify the crystalline phases using Cu Kα radiation at λ = 0.1540 nm (D8 Advance, DaVinci, BRUKER USA). The zeta potential and colloidal stability of NPs were evaluated using the dynamic light scattering (DLS) technique (SZ-100 Analyzer, Horiba Scientific, Japan). This analysis provided insights into the surface charge and colloidal stability of the NPs. For the measurement, 1 mg of α-Fe₂O₃ NPs was dispersed in 5 mL of Milli-Q water and subjected to ultrasonic irradiation for 1 min to ensure homogeneous dispersion.

2.4. In vitro drug encapsulation and release experiments

The encapsulation of lymecycline with α-Fe₂O₃ NPs was carried out by dispersing Fe₂O₃ NPs in a drug solution of 4 mg/mL for a duration of 2 h at room temperature. Upon completion of the encapsulation process, the α-Fe₂O₃ NPs were collected by centrifugation at 8000 rpm for 10 min. The absorbance of the supernatant was measured at 360 nm using UV–Vis spectroscopy. The difference in absorbance before and after the encapsulation process was used to calculate the amount of lymecycline encapsulated and to calculate the encapsulation efficiency (EE), using the following equations. The lymecycline-loaded α-Fe₂O₃ NPs (Lymecyline-α-Fe₂O₃) were subsequently washed with Milli-Q water to remove any free lymecycline present in the supernatant. The drug loaded α-Fe₂O₃ NPs were then used for subsequent release experiments [38].

Amount of drug encapsulated (D_encapsulated) = D_stock − D_supernatant

$$\mathit{EE}\%=\left(\frac{{\mathit{D}}_{\mathit{encapsulated}}}{D_{\mathit{stock}}}\right)\mathit{X}100$$

D_stock = Total drug amount in the stock solution

D_supernatant = Drug amount remaining in the supernatant after encapsulation

D_encapsulated = Amount of drug successfully encapsulated

For the released experiments, the drug-loaded NPs were dispersed in 2 mL centrifuge tubes containing either acetate (0.1 M) or PBS (0.15 M) buffer solutions. Later, the samples were incubated at 37°C under continuous shaking at 150 rpm to promote the release of lymecycline from the NPs. At predetermined time intervals, the dispersions were centrifuged at 10,000 rpm for 5 min, and the amount of lymecycline released was quantified by measuring the absorbance of the supernatant at 360 nm using UV–Vis spectroscopy. To maintain the concentration gradient and promote continuous drug release, the supernatant was periodically replaced with pre-warmed PBS or acetate buffer. The cumulative release of lymecycline was calculated by plotting the amount released as a function of time, thus providing insights into the release kinetics and efficiency of the encapsulated drug [39].

2.5. Agar-well diffusion method

The antibacterial activity was evaluated using the agar-well diffusion method against Gram-negative bacteria Escherichia coli (E. coli, ATCC 25,922) and Gram-positive bacteria Staphylococcus aureus (S. aureus, ATCC 25,923). MHA and MHB were used as the culture media for bacterial growth. Approximately 20 mL of MHA was poured into sterile petri dishes and allowed to solidify under aseptic conditions. After preparing 12 h bacterial culture, the bacterial suspension was evenly spread on the agar surface using a sterile swab. Wells with a diameter of 6 mm were punctured into the agar using a sterile cork borer. Aliquots of 100 µL of the bare α-Fe₂O₃ NP and lymecycline-α-Fe₂O₃ NP samples at various concentrations were added to the wells. Lymecycline served as a positive control, while PBS was used as a negative control. After incubating the plates at 37°C for 24 h, the antibacterial activity of the samples was assessed by measuring the diameter of the inhibition zones in millimeters (mm) [40].

2.6. Determination of MIC

The MIC of bare α-Fe₂O₃ NPs and lymecycline-α-Fe₂O₃ NPs was evaluated against E. coli and S. aureus using the broth microdilution method in 96-well plates. Serial dilutions of bare α-Fe₂O₃ NPs and lymecycline-α-Fe₂O₃ NPs were prepared in MHB at concentrations of 5, 10, 25, 50, 100, and 200 µg/mL. Each well of the 96-well plate was added with 200 µL of the dilution, followed by the addition of 10 µL of a bacterial suspension diluted to approximately 1 × 10⁸ CFU/mL. After incubating the plates at 37°C for 24 h, the bacterial growth in each well was assessed visually, and subsequently confirmed by measuring the optical density at 600 nm using a microplate reader. The MIC was defined as the lowest concentration of α-Fe₂O₃ NPs or lymecycline-α-Fe₂O₃ NPs that visibly inhibited bacterial growth. For the comparative analysis, sterile milliQ water and lymecycline were used for negative and positive control, respectively. All experiments were performed in triplicate to ensure the reproducibility and accuracy of the results [41].

2.7. Cell culture

L-929 fibroblast cells and THP-1 human leukemia monocytic cells were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. L-929 cell lines (passage number 15) were maintained in DMEM, while the THP-1 cell lines (passage number 21) were maintained in RPMI 1640 supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Cultures were incubated at 37°C in a humidified environment with 5% CO₂. The culture media was replaced every 2 days, and the experimental cells were harvested for further studies when they reached 80–90% confluence. Cell detachment was achieved through trypsinization using PBS containing 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.25% trypsin [42–44].

2.8. Alamar blue assay

L-929 and THP-1 cells were assessed for cell viability following incubation with bare α-Fe₂O₃ NPs and lymecycline-α-Fe₂O₃ NPs for 24 h using the alamar blue assay [39]. Cells were seeded into a 96-well plate at a density of 7 × 10⁵ cells per well and incubated at 37°C for 24 h to facilitate attachment. Post-incubation, the cells were treated with bare α-Fe₂O₃ NPs and lymecycline-α-Fe₂O₃ NPs at varying concentrations (0–200 µg/mL) and incubated for an additional 20 h. Subsequently, the wells were rinsed with PBS, followed by the addition of 10 μL of 1% alamar blue reagent to each well. The plate was then incubated for 4 h to allow for the metabolic reduction of resazurin. After incubation, the reagent was solubilized using DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability was assessed by quantifying the reduction of resazurin to resorufin, which reflects the metabolic activity of viable cells in response to α-Fe₂O₃ NP treatments [42].

2.9. Fluorescence microscopy analysis

The anticancer activity of α-Fe₂O₃ NPs and lymecycline-α-Fe₂O₃ NPs was investigated against L-929 and THP-1 cells using a fluorescent microscope (LUX3 FL, CytoSMART Technologies, The Netherlands) [42]. For fluorescent imaging, L-929 and THP-1 cells were treated with respective NPs at a concentration of 100 µg/mL and incubated for 24 h. Subsequently, the cells were washed with PBS and stained with 10 µL of 1% PI and AO for 15 min at 37°C under a 5% CO₂ atmosphere to visualize live and dead cell populations. Fluorescence imaging was performed using excitation wavelengths of 460 nm for AO and 525 nm for PI, respectively [38].

2.10. Glutathione peroxidase (GPx) and lipid peroxidase assay

The effects of lymecycline, α-Fe₂O₃ NPs, and lymecycline–α-Fe₂O₃ NPs on GPx activity were evaluated in THP-1 cells. For the investigation of total GPx activity, the cells were cultured under standard conditions, harvested, and treated with the respective compounds for 24 h [45]. In this assay, 125 µL of a master mix containing 1 mM EDTA, 1 mM sodium azide, 1 mM reduced glutathione (GSH), 12.5 mM PBS, and 0.25 mM NADPH, was added into the wells of a 96-well microplate. After the addition of 10 µL of the cell suspension to each well, the GPx activity was determined spectrophotometrically by measuring the decrease in absorbance at 340 nm, corresponding to the oxidation of NADPH. Furthermore, lipid peroxidation (LPO) was assessed by quantifying malondialdehyde (MDA) levels using the thiobarbituric acid (TBA) assay, which detects MDA-TBA adducts as an indicator of oxidative damage to lipids. Following the treatment, the cells were homogenized in a cold phosphate buffer (pH 7.4), and the homogenate was centrifuged at 10,000 × g for 10 min at 4°C to obtain the supernatant. An aliquot of the supernatant was mixed with a TBA reagent containing 0.67% thiobarbituric acid, 10% trichloroacetic acid, and acetic acid. The reaction mixture was heated in a boiling water bath at 95°C for 30 min to facilitate the formation of MDA-TBA adducts. After cooling to room temperature, the samples were centrifuged, and the absorbance of the supernatant was measured at 532 nm using a spectrophotometer. MDA concentrations were calculated using the molar extinction coefficient of the MDA-TBA complex and expressed as nmol of MDA per mg of protein [46,47].

2.11. Statistical analysis

All experiments were conducted in triplicate (n = 3) and repeated on three independent times to ensure reproducibility. Data are expressed as mean ± standard deviation (SD). Statistical analysis involved either one-way or two-way ANOVA with repeated measures, followed by Dunnett’s post hoc test for multiple comparisons. A p-value of <0.05 was considered statistically significant. Significance levels are denoted as ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, and not significant (ns, p > 0.05).

3. Results

Hematite (α-Fe₂O₃) NPs with a rhombus-shaped morphology were synthesized via a hydrothermal method, using ferric chloride and sodium molybdate as precursors. Sodium hydroxide was employed as the reducing agent. Under basic pH conditions, sodium molybdate facilitated the formation of a coordination complex with iron ions, which played a critical role in directing the anisotropic growth and morphology of the resulting NPs. The morphological features of the synthesized α-Fe₂O₃ NPs were characterized using FE-SEM and HR-TEM. The investigations revealed that α-Fe₂O₃ NPs possess a distinct rhombus-shaped morphology, with sizes ranging from ~80 to 150 nm (Figure 1). Notably, the observed d-spacing of 0.251 nm confirmed the (110) crystallographic plane of α-Fe₂O₃ (Figure 1(c)). The size distribution analysis obtained from FE-SEM images showed a mean particle size of ~114 nm (Figure S1, Supporting Information). Elemental composition analysis with EDS showed the presence of iron (Fe) and oxygen (O), confirming the formation of α-Fe₂O₃ NPs (Figure S2 and Table S2, Supporting Information). DLS measurements indicated a zeta potential of −21.5 mV, demonstrating good colloidal stability of the synthesized NPs. The optical absorption properties of α-Fe₂O₃ NPs were also investigated using UV–visible spectroscopy (Figure 2(a)). The characteristic absorption peaks observed in the range between 300 and 600 nm confirm the successful formation of α-Fe₂O₃ NPs. The strong absorption peak centered at 300 nm corresponds to a direct ligand-to-metal charge transfer transition, involving non-bonding oxygen 2p orbitals and d–d electronic transitions associated with Fe³⁺ ions. Additionally, α-Fe₂O₃ NPs were investigated by FTIR spectroscopy to gain more detailed information about their composition and functional groups present on their surface (Figure 2(b)). The FTIR spectrum of α-Fe₂O₃ NPs exhibited distinct bands corresponding to various functional groups and vibrational modes. A prominent absorption peak at 547 cm⁻¹ was assigned to the Fe–O stretching vibration (i.e., characteristic of metal–oxide bonds). Additional peaks observed at 892, 947, and 1153 cm⁻¹ were attributed to C–H stretching, in-plane bending (δ), and C–N vibrational modes, respectively. Furthermore, other peaks appeared at 1534, 1640, and 1746 cm⁻¹ are attributed to the stretching vibrations of carboxylate groups (–COO⁻), amine groups, and carbonyl groups (–C=O), respectively. The absorption bands at 3403 and 1415 cm⁻¹ were indicative of O–H stretching vibrations, confirming the presence of hydroxyl groups on the nanoparticle surface.

Figure 1.

Morphological investigation of α-Fe₂O₃ NPs. (a) FE-SEM image showing the rhombus-shaped morphology of α-Fe₂O₃ NPs, and HR-TEM images of the same NPs at low (b) and high (c) magnifications to show their morphological and crystallographic features.

Figure 2.

(a) UV- Visible, (b) FTIR spectrum, (c) XRD spectrum, and (d) Raman spectrum of synthesized α-Fe₂O₃ NPs.

To further investigate the crystal structure of α-Fe₂O₃ NPs, XRD analysis was performed. Figure 2(c) displays the XRD pattern of the α-Fe₂O₃ NPs, with diffraction peaks indexed according to the standard Joint Committee on Powder Diffraction Standards (JCPDS) card No. 01–089–0598. The spectrum displays sharp crystalline peaks corresponding to the (012), (104), (110), (113), (024), (116), (211), (214), and (300) crystallographic planes of α-Fe₂O₃ at 2θ values of 23.0°, 32.8°, 35.6°, 40.7°, 49.2°, 53.8°, 56.7°, 62.2°, and 63.8°, respectively [48]. These peaks confirm the successful synthesis of crystalline α-Fe₂O₃ NPs, indicating a well-defined crystal structure and high crystallinity. The Raman spectrum of the as-prepared α-Fe₂O₃ NPs exhibited distinct and well-resolved peaks at 287, 401, 489, 603, and 650 cm⁻¹, which are corresponding to the characteristic vibrational modes associated with the crystalline hematite phase [49]. While the peak at 287 cm⁻¹ can be assigned to the Eg mode that reflects the in-plane bending vibration of Fe–O bonds, the peak at 401 cm⁻¹ corresponds to the B2g mode associated with the vibration of oxygen atoms within the crystal lattice [19,50]. Additionally, the peaks at 489, 603, and 650 cm⁻¹ are attributed to the A1g mode representing the symmetric stretching vibrations of the Fe–O bonds in the hematite crystal structure, as shown in Figure 2(d). These peaks match well with the previously reported characteristics of α-Fe₂O₃, thereby confirming the successful synthesis of crystalline hematite NPs.

The encapsulation efficiency of lymecycline within the α-Fe₂O₃ NPs was determined to be approximately 30%, corresponding to 2.4 mg of drug encapsulated in 8 mg of NPs. In vitro drug release studies demonstrated an initial linear release profile of lymecycline during the first 6 h, followed by a significant decrease in the release rate thereafter, under both acidic (pH 5.5) and physiological (pH 7.4) conditions. After 24 h, the cumulative drug release reached approximately 60% at pH 5.5 and 43% at pH 7.4, as illustrated in Figure 3. These findings indicate a pH-responsive drug release behavior, with a higher release rate observed under mildly acidic conditions, thereby supporting the potential of α-Fe₂O₃ NPs for targeted drug delivery in environments such as tumor tissues.

Figure 3.

Drug release kinetics of lymecycline-loaded α-Fe₂O₃ NPs as a function of time.

The MIC assay confirmed that a concentration of 50 µg/mL of lymecycline-α-Fe₂O₃ NPs was sufficient to effectively inhibit the growth of both bacterial strains, as summarized in Table 1 and illustrated in Figure 4(c,d). Additionally, the zones of inhibition were measured for both bare and lymecycline-α-Fe₂O₃ NPs against E. coli and S. aureus. For E. coli, the maximum zone of inhibition was 31 ± 0.5 mm for lymecycline-α-Fe₂O₃ NPs at 100 µg/mL. For S. aureus, the zone of inhibition was 23 ± 1.5 mm at the same concentration, while bare α-Fe₂O₃ NPs showed no zone of inhibition for either bacterial strain, indicating negligible intrinsic antibacterial activity as shown in Table 2 and Table S1, Supporting information, and Figure 4 and Figure S4, Supporting information. Separately, the antibacterial efficacy of lymecycline (free drug), bare α-Fe₂O₃ NPs, and lymecycline–α-Fe₂O₃ NPs was also evaluated at concentrations of 50 and 100 µg/mL against E. coli and S. aureus via the broth microdilution method. At 50 µg/mL, lymecycline exhibited inhibitory activity, achieving 52% inhibition against E. coli and 35% against S. aureus. In contrast, bare α-Fe₂O₃ NPs demonstrated negligible antibacterial activity at the same concentration, with inhibition rates of only 5% and 3%, respectively. However, the antibacterial activity of the bare NPs increased moderately at 100 µg/mL, yielding 9% inhibition for E. coli and 15% for S. aureus. On the other hand, the lymecycline–α-Fe₂O₃ NPs exhibited slightly lower inhibition than lymecycline alone at 50 µg/mL while outperforming the free drug at 100 µg/mL. Notably, it showed inhibition rates of 67% for E. coli and 66% for S. aureus at 100 µg/mL. Thus, these results suggest a concentration-dependent synergistic effect between lymecycline and α-Fe₂O₃, enhancing antibacterial efficacy at higher doses.

Table 1.

The table shows the MIC values for lymecycline, α-Fe₂O₃ and lymecycline-α-Fe₂O₃ NPs against E. coli and S. aureus. These values are the lowest concentrations of each drug that effectively inhibit bacterial growth under standardized conditions.

	E. coli		S. aureus
	Concentration (µg/mL)	Cell inhibition (%)	Concentration (µg/mL)	Cell inhibition (%)
Control (PBS)	0	0	0	0
Lymecycline	50	66	50	64
α-Fe2O3	50	5	50	3
	100	9	100	15
Lymecycline-α- Fe2O3	50	51	50	35
	100	67	100	66

Figure 4.

Evaluation of agar-well diffusion assay, and MIC of lymecycline, α-Fe₂O₃, and lymecycline-α- Fe₂O₃ NPs against E. coli and S. aureus. The zone of inhibition against E. coli (a) and S. aureus (b), and the cell viability percentages for E. coli (c) and S. aureus (d). The broth dilution assay data represented as the mean ± standard deviation (SD) from three different experiments, and the significance difference was set to P < 0.05 when compared with the control by one-way ANOVA followed by Dunnett’s multiple comparison method. A significant difference indicated by ****corresponds to a P < 0.0001, ***corresponds to P < 0.001, **corresponds to P < 0.01, *corresponds to P < 0.1 and non-significant (ns).

Table 2.

The table shows the inhibition zone diameters for lymecycline, α-Fe₂O₃, and lymecycline-α- Fe₂O₃ NPs against E. coli and S. aureus..

	E. coli (mm)	S. aureus (mm)
Control	No Zone	No Zone
Antibiotics	41 ± 1.0	34 ± 1.0
α-Fe2O3	No Zone	No Zone
lymecycline-α- Fe2O3	31 ± 0.5	23 ± 1.5

The anticancer potential of bare α-Fe₂O₃ NPs and lymecycline-loaded α-Fe₂O₃ NPs was evaluated by measuring cell viability in human monocytic leukemia (THP-1) cells, alongside cytocompatibility studies in normal fibroblast (L-929) cells (Figure 5, and Figures S5 and S6, Supporting Information). In THP-1 cells, bare α-Fe₂O₃ NPs exhibited 38% and 40% cell death at 100 µg/mL and 200 µg/mL, respectively. In contrast, the lymecycline-loaded α-Fe₂O₃ NPs showed significantly high cytotoxicity of 52% and 72% against THP-1 cells at 100 µg/mL and 200 µg/mL, respectively. In the case of L-929 cells, bare α-Fe₂O₃ NPs demonstrated excellent biocompatibility, maintaining high cell viability at 89% and 84% for 100 µg/mL and 200 µg/mL, respectively. Similarly, the lymecycline-loaded α-Fe₂O₃ NPs maintained considerable cell viability, with values of 83% at 100 µg/mL and 73% at 200 µg/mL, thus indicating selective cytotoxicity toward cancerous cells while maintaining acceptable biocompatibility in normal cells.

Figure 5.

The biocompatibility and anticancer activity investigations of α-Fe₂O₃ NPs, lymecycline free drug, and lymecycline- α-Fe₂O₃ NPs against normal L-929 fibroblast cells and THP-1 cancer cells. The cell viability of α-Fe₂O₃ NPs (a), and lymecycline-α-Fe₂O₃ NPs (b) against THP-1 cancer cells. The cell viability of α-Fe₂O₃ NPs (c), and lymecycline-α-Fe₂O₃ NPs (d) against L-929 fibroblast cells. The cell viability data is shown as the mean ± standard deviation (SD) from three different experiments, and the significance was set to P < 0.05 when compared with the control, by one-way ANOVA followed by Dunnett's multiple comparison method. A significant difference indicated by ****corresponds to a P < 0.0001, ***corresponds to P < 0.001, **corresponds to P < 0.01, *corresponds to P < 0.1 and non-significant (ns).

The GPx activity assay revealed a significant elevation in ROS-mediated antioxidant response in THP-1 cells treated with α-Fe₂O₃ and lymecycline-loaded α-Fe₂O₃ NPs. Notably, the optical density (OD₃₄₀ nm) values recorded for α-Fe₂O₃ and lymecycline-α-Fe₂O₃ NP-treated cells were 0.072 and 0.064 per 10⁶ cells/mL, respectively, both markedly higher than the control group (0.026 per 10⁶ cells/mL). In contrast, treatment with free lymecycline (0.037 per 10⁶ cells/mL) did not show a statistically significant difference from the control group (Figure S7, Supporting Information). Lipid peroxidation (LPO) in THP-1 cells was also assessed using the fluorescent DPPP dye. After 24 h of treatment, a concentration-dependent increase in fluorescence intensity was observed across all groups, indicating progressive oxidative damage (Figure S8A and S8B, Supporting Information). While α-Fe₂O₃-treated group showed a substantial rise in DPPP fluorescence and reflected enhanced oxidative stress, the lymecycline-α-Fe₂O₃ group exhibited the highest fluorescence intensity among all tested groups. This suggests an amplified ROS-mediated peroxidative effect in the presence of hybrid NPs/drug conjugate. MDA levels, a key biomarker of lipid peroxidation and cellular damage, were significantly elevated in the α-Fe₂O₃ and lymecycline-α-Fe₂O₃-treated cells, showing 2.82-fold and 3.24-fold increase, respectively, compared to the control group. Conversely, free lymecycline treatment did not result in a significant change in MDA levels, aligning with its minimal effect on oxidative stress markers (Figure S8C, Supporting Information).

4. Discussion

Iron oxides represent a vital class of transition metal oxides with considerable significance in biomedical science. Among the various crystalline forms, hematite (α-Fe₂O₃) is the most stable oxide under ambient conditions. In the present study, rhombus-shaped α-Fe₂O₃ NPs were successfully synthesized using ferric chloride and sodium molybdate as precursors, under basic pH conditions. It is believed that the synthesis kinetics are significantly influenced by the presence of sodium molybdate, which played a dual role in the reaction system. First, it served as a morphology-directing agent, coordinating with ferric ions to form transient complexes that modulate the rate of nucleation and anisotropic growth [51]. Second, molybdate species promoted a controlled hydrolysis of Fe³⁺ ions, slowing down the overall reaction rate and allowing for uniform nucleation followed by oriented crystal growth under alkaline pH conditions. This modulation of the reaction kinetics is critical in achieving the rhombus-shaped α-Fe₂O₃ NPs. Previous investigations demonstrate that a slower nucleation rate, followed by selective growth along specific crystallographic planes, leads to anisotropic structures rather than a spherical morphology [52–54]. Furthermore, the alkaline condition also plays a key role in the formation of iron hydroxide intermediates, which dehydrate and rearrange into α-Fe₂O₃ nuclei. The presence of molybdate ions possibly adsorbs preferentially on specific facets, inhibiting growth along those planes and thereby promoting rhombic crystal development.

The FE-SEM and HR-TEM investigations revealed that the synthesized α-Fe₂O₃ NPs exhibited a well-defined rhombus-like morphology, with particle sizes ranging between 80 and 150 nm (Figure 1). The lattice fringe spacing of 0.251 nm corresponds to the (110) crystallographic plane of α-Fe₂O₃, thereby confirming the crystalline nature of the formed NPs (Figure 1(c)). Furthermore, the EDS spectrum also shows the presence of iron (Fe) and oxygen (O) atoms, revealing the successful formation α-Fe₂O₃ NPs. The UV–visible spectral analysis provided crucial insights into the electronic structure and optical behavior of the synthesized α-Fe₂O₃ NPs. The broad absorption in the 300–600 nm range and the distinct peak at ~300 nm are consistent with the previously reported optical characteristics of hematite nanostructures, thereby confirming the phase purity and formation of the α-Fe₂O₃ crystalline phase. Notably, the strong absorption peak at 300 nm is particularly significant, as it indicates ligand-to-metal charge transfer and Fe³⁺ d–d transitions, showcasing the semiconducting nature of the formed NPs.

FTIR analysis revealed the presence of various surface functional groups such as hydroxyl (−OH), carboxylate (−COO⁻), amine (−NH₂), and carbonyl (C=O). These groups likely originate from precursor decomposition and residual organic moieties and are crucial in determining surface reactivity, colloidal stability, and encapsulation efficiency. To gain more detailed information about the structural integrity, crystal structure, and phase composition, the synthesized α-Fe₂O₃ NPs were investigated by XRD and Raman spectroscopy. The XRD analysis revealed a series of sharp and intense diffraction peaks corresponding to the (012), (104), (110), (113), (024), (116), (211), (214), and (300) planes, matching well with standard reference data. The absence of secondary phases or amorphous features along with narrow peak widths in the XRD pattern confirms that the product formed is a highly crystalline and pure hematite phase (α-Fe₂O₃). The Raman spectrum also exhibited distinct and well-resolved peaks at 287, 401, 489, 603, and 650 cm⁻¹, which are characteristic of the hematite polymorph of Fe₂O₃ [49]. Thus, these findings demonstrate that the synthesis kinetics, influenced by the chemical environment and molybdate coordination, play a decisive role in tailoring the morphology, crystallinity, and surface functionality of α-Fe₂O₃ NPs. Such engineered NPs have great potential for applications such as targeted drug delivery, antibacterial coatings, and cancer therapy.

The encapsulation efficiency of lymecycline (~30%) indicates a moderate loading onto the α-Fe₂O₃ nanoparticle matrix, most likely sufficient for anticancer therapy and antibacterial applications. The observed pH-sensitive-release behavior is advantageous as the nanocarriers can preferentially release the drug in acidic microenvironments such as cancerous tissues, intracellular endosomes, and inflammatory sites while maintaining relative stability in the bloodstream. The initial burst release phase may offer immediate therapeutic action, whereas the subsequent sustained release is useful for prolonged drug availability and reducing the need for frequent dosing. This dual-phase profile enhances the efficacy and safety profile of the nanocarrier system, minimizing systemic side effects and maximizing localized drug action. Thus, these findings collectively support the suitability of α-Fe₂O₃ NPs as pH-responsive drug delivery vehicles for anticancer therapy and antibacterial applications.

The antibacterial studies conducted on the synthesized NPs clearly demonstrate the limited efficacy of bare α-Fe₂O₃ NPs when used alone, with minimal inhibition observed against both E. coli and S. aureus. This suggests that the intrinsic antibacterial properties of α-Fe₂O₃ are negligible and that these NPs are not sufficient for significant microbial inhibition in the absence of antibacterial drugs. However, the antibacterial activity was significantly improved after encapsulation with lymecycline. Furthermore, the lymecycline–α-Fe₂O₃ NPs exhibited substantially higher inhibition rates compared to the free drug, especially at a concentration of 100 µg/mL. Specifically, they exhibited 66% inhibition against S. aureus and 67% against E. coli, surpassing the efficacy of free lymecycline at the same concentration. This suggests a potential synergistic or additive effect between the drug and the nanoparticle matrix at higher doses. At concentrations 50 µg/mL, lymecycline free drug showed greater inhibition than the nanoformulation, while the encapsulated form still maintained appreciable antibacterial activity. This observation of concentration-dependent synergistic effect between lymecycline and α-Fe₂O₃ was further supported by the MIC assay. These results confirmed the delayed or controlled-release behavior of the drug from the nanoparticle matrix, thus allowing sustained interaction with bacterial cells over time. Previous investigation on tetracycline encapsulated in gold decorated ZnO NPs demonstrated that dual sensitization and energy transfer mechanisms played a key role in improving the antibacterial activity [17]. This system produced high ROS levels under white light exposure that resulted in effective targeting of Staphylococcus hominis while reducing antibiotic residues to limit resistance development. It is worth noting that the study only focused on the antibacterial activity of Gram-positive bacteria. In a separate study, the IONPs encapsulated with tetracycline hydrochloride showed inhibition zones of 33.3 ± 2.0 mm for E. coli and 36.5 ± 0.7 mm for S. aureus in N,N-dimethylformamide [55]. Notably, the use of N,N-dimethylformamide solvent may contribute to antibacterial effects and thereby can influence in vivo outcomes. However, the present study with solvent-free lymecycline formulations demonstrates potentially greater antibacterial efficacy. In summary, the proposed lymecycline–α-Fe₂O₃ NPs demonstrated synergistic antibacterial activity, most likely resulting from the combined bacterial disruption effects of α-Fe₂O₃ and the potent inhibition of protein synthesis by lymecycline.

The cytotoxicity results demonstrate the promising anticancer potential of α-Fe₂O₃ NPs, particularly when encapsulated with lymecycline. While bare α-Fe₂O₃ NPs displayed moderate toxicity toward THP-1 leukemia cells, the lymecycline-loaded α-Fe₂O₃ NPs exhibited markedly enhanced cytotoxicity of up to 72% at an NP concentration of 200 µg/mL. This enhancement may be attributed to the synergistic interaction between the inherent properties of α-Fe₂O₃ and the chemotherapeutic activity of lymecycline, most likely facilitated by the increased cellular uptake or influencing the apoptosis pathways in malignant cells. Importantly, both nanoparticle formulations showed high compatibility with L-929 normal fibroblast cells, with viability remaining above 73% even at elevated concentrations. The observed selective cytotoxicity of lymecycline-loaded α-Fe₂O₃ NPs toward cancer cells can be primarily attributed to their ability to catalyze Fenton reactions, resulting in the generation of ROS. Separately, a similar investigation of tetracycline-silver (TC-Ag) nanoparticle system on MCF-7 breast cancer cells exhibited a dose-dependent cytotoxicity of 60% at 50 µg/mL, which increased to 85% at 100 µg/mL after 24 h [56]. Previous reports on tetracycline-loaded ferrite NPs (e.g., Tet/Ni₀.₅Co₀.₅Fe₂O₄/SiO₂ and methionine-coated NiFe₂O₄) show multifaceted mechanisms ranging from ROS generation to biochemical pathway disruption for the enhanced anticancer efficacy of iron oxide-based nanoplatforms [13,57]. Furthermore, α-Fe₂O₃ core not only serves as a targeted delivery system for lymecycline but also releases iron ions and disrupts key cellular antioxidant defenses such as the glutathione peroxidase 4 (GPX4) pathway to sensitize cancer cells to oxidative damage. Notably, the observed elevation of intracellular ROS levels and lipid peroxidation markers in THP-1 cells treated with both bare α-Fe₂O₃ and lymecycline-loaded α-Fe₂O₃ NPs suggests the possible role of the ferroptosis process in the anti-cancer activity. The GPx assay exhibited a significant increase in GPx activity and MDA levels along with intensified lipid peroxidase fluorescence, indicating oxidative stress-mediated cellular damage. Furthermore, the overproduction of ROS likely initiates peroxidation of membrane lipids, and culminating in irreversible disruption of cellular integrity, i.e., a clear sign of ferroptosis. Such a mechanism offers a promising therapeutic avenue, as ferroptosis circumvents resistance pathways associated with apoptosis [58–60]. Previous studies on piezoelectric Fe/BaTiO₃-based nanostructures have demonstrated a synergistic approach to cancer therapy through controllable ferroptosis, immune activation, and CD8⁺ T cell-mediated responses. It is worth noting that the full immunogenic implications of ferroptotic death remain to be fully elucidated [18]. Thus, the current findings emphasize the potential of iron oxide–based nanoplatforms not only to directly kill cancer cells via ferroptosis but also to possibly induce immune responses, offering a dual modality for cancer treatment.

Despite promising in vitro results, this study has notable limitations. The experiments were conducted in simplified cell culture models, which deviates significantly from the complex in vivo environment. The role of factors such as immune interactions, nanoparticle biodistribution, metabolism, and clearance remains unexplored. Additionally, drug concentrations effective in in-vitro conditions may not directly translate to therapeutic levels in vivo due to differences in absorption and tissue penetration. To address these gaps, future studies will focus on in vivo evaluations, advanced 3D culture systems, and formulation optimization to improve stability, targeted delivery, and controlled drug release. These efforts are essential for advancing iron oxide nanoparticle-based therapies toward clinical applications.

5. Conclusions

We have reported rhombus-shaped α-Fe₂O₃ NPs with a size range of ~80 to 150 nm in length and a width of ~50 nm via hydrothermal method, and demonstrated their potential for antibacterial and anti-cancer applications. The developed lymecycline-loaded α-Fe₂O₃ NPs demonstrated a drug encapsulation efficiency of approximately 30%. The release kinetics demonstrated a pH-dependent release of lymecycline with cumulative release reaching up to 60% at pH 5.5 and 43% at pH 7.4. These nanocarriers showed potent cytotoxicity against THP-1 leukemia cells while maintaining favorable biocompatibility with normal L-929 fibroblast cells. The cytotoxicity against cancer cells is possibly driven by α-Fe₂O₃-induced overproduction of ROS through ferroptosis that led to lipid peroxidation, oxidative stress, and irreversible membrane damage. In addition to their anticancer activity, the lymecycline-α-Fe₂O₃ NPs displayed strong antibacterial efficacy against E. coli and S. aureus. The antibacterial mechanism is likely attributed to the sustained release of lymecycline, which disrupts bacterial protein synthesis and impairs essential metabolic pathways, thereby inhibiting microbial proliferation. Overall, the lymecycline-α-Fe₂O₃ NPs demonstrate dual functionality with potent anticancer and antibacterial activities, highlighting their significant potential as a versatile nanoplatform for therapeutic applications and future drug development.

Funding Statement

Dr. Anandhakumar Sundaramurthy acknowledges the financial support from Anusandhan National Research Foundation (ANRF), New Delhi, Government of India [File no. CRG/2020/004175].

Article highlights

• Rhombus-shaped α-Fe₂O₃ nanoparticles (80–150 nm length, ~50 nm width) were synthesized via novel hydrothermal method.

• The α-Fe₂O₃ NPs showed 30% encapsulation efficiency with lymecycline.

• Lymecycline release from α-Fe₂O₃ NPs was pH-responsive, with 60% release at pH 5.5 and 43% at pH 7.4.

• Lymecycline-loaded α-Fe₂O₃ NPs exhibited enhanced cytotoxicity (72%) against THP-1 leukemia cancer cells.

• Biocompatibility assays confirmed the biocompatibility of bare and drug-loaded α-Fe₂O₃ NPs against L-929 fibroblast cells.

• Antibacterial assay showed significant inhibition of E. coli and S. aureus.

• Anticancer effect is likely arising from ferroptosis, an iron-dependent cell death pathway triggered by iron accumulation from α-Fe₂O₃ NPs.

Author contributions

Sakshi Bajhal: Writing – Original Draft, Methodology, Formal Analysis. Nishakavya Saravanan: Writing – Original Draft, Methodology, Formal analysis. Anandhakumar Sundaramurthy: Writing – Review & Editing, Funding Acquisition, and Conceptualization.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing assistance

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosure

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435889.2025.2542716