Bioinspired 8‑hydroxyquinoline-Fe₃O₄ nanostructures from Citrullus colocynthis exhibit strong antibacterial, antifungal, and anticancer effects

Abstract

This study presents a novel green-synthesized iron oxide nanostructure (ION) using Citrullus colocynthis extract (CCE) and functionalized with 8-hydroxyquinoline (8HQ) to enhance antibacterial, antifungal, and anticancer activities. IONs were synthesized via CCE-mediated reduction or chemical coprecipitation, coated with 8HQ, and characterized by FE-SEM, EDAX, and FT-IR. The antimicrobial efficacy was assessed against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Candida albicans through CLSI-guided MIC/MBC assays, while cytotoxicity was evaluated on MCF7 and Hep-G2 cancer cells using MTT assays. Results demonstrated that CCE-ION exhibited enhanced antimicrobial activity compared to chemically synthesized ION. The MIC values for CCE-ION were 125 µg/mL for P. aeruginosa, 250 µg/mL for E. coli, 500 µg/mL for S. aureus and C. albicans, and 1000 µg/mL for E. faecalis, with corresponding MBC values of 250, 250, 500, 500, and 1000 µg/mL, respectively, indicating strong bactericidal and fungicidal properties. FE-SEM analysis confirmed spherical nanoparticles (35–40 nm) with balanced iron oxide and organic composition. The 8HQ@CCE-ION nanocomposite exhibited significant cytotoxicity, with IC₅₀ values of ≈ 489 µg/mL against MCF-7 cells and ≈ 183 µg/mL against Hep-G2 cells (compared to ≈ > 500 µg/mL and ≈ 450 µg/mL for free CCE, respectively), achieving > 80% cell death at 250 µg/mL and highlighting enhanced cellular uptake and synergistic effects. This eco-friendly nanoplatform successfully integrates CCE’s natural bioactivity with 8HQ’s metal-chelating properties, achieving significant pathogen viability reduction at MIC doses while offering promising anticancer potential. The dual-functional approach positions 8HQ@CCE-ION as a potential therapeutic candidate for drug-resistant infections and cancer treatment.

Introduction

Microbial and fungal infections are among the most common and serious human diseases, and extensive research has been conducted to detect, treat, and prevent these pathogens¹. Bacterial strains such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, as well as the yeast Candida albicans, are responsible for various infections that affect human health and quality of life². Although many potent antifungal and antibacterial drugs are currently available to combat these infections^3,4, the emergence of resistant strains, the genetic diversity of microbial pathogens, and the adverse effects of these drugs pose significant challenges to the development of novel and effective therapies^5,6.

Metal nanoparticles are promising alternatives to conventional drugs, as they have shown remarkable antimicrobial and anticancer activities without the drawbacks of chemical pharmaceuticals^7–9. Various methods, such as physical, chemical, and biological processes, can synthesize metal nanoparticles. Among these methods, green synthesis of metal nanoparticles using plant extracts has attracted considerable attention, as it offers several advantages over other methods^9–11. Green synthesis of metal nanoparticles is environmentally friendly, as it avoids using toxic and hazardous chemicals, such as sodium borohydride, as reducing agents¹¹. It is also cost-effective, using renewable and abundant plant resources as reducing and stabilizing agents¹². Moreover, it is biocompatible, as it does not involve any harmful chemicals that may interfere with the biological and pharmaceutical applications of the nanoparticles¹². Several studies have reported the green synthesis of iron nanoparticles using various plant extracts and their antimicrobial and anticancer properties^13–15.

Citrullus colocynthis (CC), commonly known as bitter apple or colocynth, is a valuable medicinal plant in arid and desert regions. It is indigenous to the Mediterranean region and Asia¹⁶. This plant has a history of traditional therapeutic use for treating gastrointestinal diseases and musculoskeletal disorders and as an anti-inflammatory, larvicidal, antifungal, and antibacterial agent¹⁷. The fruit of CC contains several bioactive compounds, including glycosides, flavonoids, alkaloids, carbohydrates, fatty acids, and essential oils¹⁷. Phytochemical analysis of this plant has revealed the presence of various bioactive compounds, such as pyridine and phenolic acids. Notably, cucurbitacins have been identified as the primary components of CC fruits¹⁸. These compounds are responsible for the pharmacological activities of this plant, such as anti-diabetic, anti-hyperlipidemic, and anticancer effects¹⁷. However, despite the reported benefits of this plant, few studies have investigated the effect of its extract on the green synthesis of iron nanoparticles and their antimicrobial and cytotoxic potential¹⁹. Therefore, the main objective of this study is to explore the novel application of this plant extract as a reducing and stabilizing agent for the green synthesis of iron nanoparticles and to evaluate their antibacterial, antifungal, and anticancer activities.

Despite advancements in green synthesis of iron oxide nanoparticles (IONs) using plant extracts like C. colocynthis for antimicrobial^20–23 or anticancer applications²⁴, and the known metal-chelating and apoptotic properties of 8-hydroxyquinoline (8HQ)²⁵, a critical research gap remains, including the lack of integrated platforms combining these elements into a single, eco-friendly nanocomposite for dual-functional therapy. Recent studies highlight IONs’ potential in targeted drug delivery and oxidative stress induction^26–31, yet none have explored C. colocynthis extract (CCE) as a reducing/stabilizing agent for 8HQ-functionalized IONs (8HQ@CCE-ION) to synergistically enhance efficacy against drug-resistant pathogens and cancer cells while minimizing toxicity. This study addresses this gap by developing and evaluating such a novel nanoplatform, offering a sustainable alternative to conventional chemical syntheses. In this study, we have developed a novel nanomedicine delivery platform by coating 8HQ on the green-synthesized ION using CCE. 8HQ is a metal chelator reported to have anticancer activity by inducing apoptosis and inhibiting angiogenesis²⁵. By integrating 8HQ with ION, we aimed to improve the nanomedicine’s antibacterial, antifungal, and anticancer efficacy and reduce CCE toxicity. We have characterized the synthesized nanoparticles using techniques such as FT-IR, FE-SEM, and EDAX. Using the broth micro-dilution method, we have additionally tested the nanoparticles for their antibacterial and antifungal activity against various Gram-positive and Gram-negative bacteria and yeast. In addition, we have evaluated the cytotoxic and antitumor activity of the nanoparticles against the breast cancer cell line (MCF7) and human liver cancer cell line (Hep-G2) by MTT assay. The results of this study show the potential of this nanoplatform as a biocompatible, safe, and efficient nanomedicine for cancer therapy and infection control.

Materials and methods

Reagents and materials

All reagents used in this study were of analytical grade and purchased from Merck & CO. These included iron (III) chloride hexahydrate (FeCl₃.6H₂O), iron (II) sulfate heptahydrate (FeSO₄.7H₂O), 8-hydroxyquinoline, aqueous ammonia (NH₃.H₂O), ethanol (C₂H₅OH), and methanol (CH₃OH). The CC fruit was purchased from the local market of Shiraz, Fars province, Iran. The plant material was formally identified and authenticated by a botanist (Mrs Sedigheh Khademian) at the Department of Pharmacognosy, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran. A voucher specimen of the plant material has been deposited in the herbarium of Shiraz University of Medical Sciences under the accession number SUMS/232.1402.

Extraction of plant

The process commenced with the cleansing of CC fruits, first with tap water and subsequently with distilled water. Following this, the seeds and pulp (mesocarp) were meticulously gathered subsequent to air drying. Approximately 20 g of seeds underwent desiccation in a hot oven set at 70 °C, after which they were finely pulverized utilizing a commercial grinder and sifted through 80 mesh sieves. The resulting finely ground material was then stored in polythene bags and refrigerated for further analysis.

The initial crude extraction phase involved agitating 10-gram samples in an orbital shaker for a duration of 8 to 9 h, employing a 90% ethanol/methanol (v/v) solution within a 250 mL beaker at ambient temperature for 24 h. The residue underwent a subsequent re-extraction process using an additional 90% ethanol/methanol solution for another 24-hour cycle under identical conditions. Post-extraction, the filtrate was separated, and the solvent was eliminated via evaporation utilizing a rotary evaporator under reduced pressure (vacuum). The resulting dehydrated CCE was then stored at 40 °C in a refrigerator until required for further analysis. Extract yields were determined through gravimetric assessment, and the semisolid crude extracts were meticulously transferred quantitatively to the corresponding extraction solvent for preservation.

The percentage yield of the CCE, represented in terms of dry weight (DW), was computed using the following formula:

Preparation of chemically and green synthesized 8HQ@ION

In this study, two types of 8HQ@ION named chemically synthesized one (8HQ@ION) and green synthesized (8HQ@CCE-ION) were prepared.

To initiate the preparation of chemically synthesized 8HQ@ION, 3.89 g (14 mmol) of FeSO₄·7H₂O and 4.55 g (28 mmol) of FeCl₃·6H₂O were combined in a 1 L round-bottom flask containing 320 mL deionized water (DI). The resulting mixture was agitated for 30 min at 80 °C, yielding a homogeneous suspension.

In contrast to the original procedure, the use of concentrated CCE (200 mL) instead of 320 mL of DI water was implemented to synthesize green synthetized ION.

Subsequently, for both chemically and green synthesized ION, 0.1 g of 8HQ was introduced into the suspension, and the reaction was sustained under constant stirring for an additional 30 min. Following this, 30 mL of 25% NH₃ solution was carefully added dropwise to the suspension using a burette, and the reaction was allowed to proceed for an additional hour under the same conditions.

Upon completion of the synthesis, a robust magnet was used to isolate the 8HQ@ION and 8HQ@CCE-ION, which were then subjected to three successive washes with deionized water to neutralize any residual magnetite nanoparticles. Subsequently, the resulting nanoparticles were dried in an oven for two hour at 60 °C.

Characterization of nanoparticles

The nanoparticles (ION, CCE-ION, 8HQ@ION, and 8HQ@CCE-ION) were characterized by various techniques to determine their size, morphology, elemental composition, and crystalline structure.

The morphology and elemental mapping of the nanoparticles were obtained by field emission scanning electron microscopy (FE-SEM) using a Tescan model Mira III instrument (Tescan, Czech Republic) equipped with an energy-dispersive X-ray spectroscopy (EDX) detector (S Max, Oxford Instruments, UK). The samples were coated with a thin layer of gold to improve the conductivity and prevent charging. The FE-SEM images were taken at different magnifications and accelerating voltages. The EDX mapping was performed to visualize the spatial distribution of the nanoparticle elements. The EDX spectra were also recorded to quantify the elemental composition of the nanoparticles. The EDX data were analyzed using the INCA software (Oxford Instruments, UK).

X-ray Diffraction (XRD) Analysis: Crystalline structure and phase identification were performed using a Philips X’Pert MPD diffractometer with Cu Kα radiation (λ = 1.5406 Å) at 40 kV and 30 mA. Powdered samples were scanned over a 2θ range of 10–80° with a step size of 0.02° and a scan speed of 1°/min. The obtained diffraction patterns were analyzed using X’Pert HighScore software and compared to standard reference data for magnetite (Fe₃O₄), specifically JCPDS card No. 19–0629. This comparison confirmed characteristic peaks at 2θ values of approximately 30.1° (220), 35.5° (311), 43.1° (400), 53.4° (422), 57.0° (511), and 62.6° (440), indicative of a pure cubic spinel structure without detectable impurities. Crystallite size was estimated using the Scherrer equation: D = (Kλ)/(β cos θ), where D is the crystallite size, K is the shape factor (0.9), λ is the X-ray wavelength, β is the full width at half maximum (FWHM) of the (311) peak, and θ is the Bragg angle, yielding an average size of ~ 35 nm consistent with FE-SEM observations.

The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the nanoparticles were measured using a Zetasizer Nano ZS (Malvern Panalytical, UK) equipped with a 633 nm He-Ne laser. For analysis, each nanoparticle sample was dispersed in phosphate-buffered saline (PBS, pH 7.4) at 0.1 mg/mL and sonicated for 5 min to ensure homogeneity. The hydrodynamic size and PDI were determined using dynamic light scattering (DLS) at a back-scattering angle of 173° at 25 °C. Zeta potential was assessed through laser Doppler velocimetry in a folded capillary cell (DTS1070). All measurements were performed in triplicate, and the results are presented as the mean ± standard deviation (SD).

The morphology and core size of the nanoparticles were examined using Transmission Electron Microscopy (TEM) with a JEOL JEM-1400Plus operated at 120 kV. For the TEM analysis, a drop of a dilute aqueous dispersion of nanoparticles (0.01 mg/mL) was placed on a carbon-coated copper grid and allowed to air dry. The core diameter of the nanoparticles was determined by manually measuring over 100 individual particles from multiple TEM images, using ImageJ software (National Institutes of Health, USA). The results are presented as the mean diameter with standard deviation.

Antibacterial and antifungal activity

Determination of minimum inhibitory concentration (MIC)

The antibacterial susceptibility of the samples was evaluated according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI)³. The minimum inhibitory concentrations (MICs) of the compounds were determined by the microdilution broth assay, as described by Pansara et, al³². The effect of the compounds on the growth rate of five microorganisms, namely P. aeruginosa, E. faecalis, E. coli, S. aureus, and C. albicans, was studied using brain-heart infusion (BHI) broth. The microorganisms were obtained from the Persian Type Culture Collection (PTCC) and cultured according to the standard protocols.

The microdilution broth assay was performed as follows: All 96 microtiter plate wells were filled with 90 µL of BHI broth. Then, 90 µL of the compounds were added to the first row of wells, resulting in a 1000 µg/mL concentration. Two-fold serial dilutions of the compounds were made by transferring 90 µL from the first row to the second row, and so on, until the seventh row, resulting in a final concentration of 7.8 µg/mL. The eighth row, containing only BHI broth, was used as a negative control. A total of 10 µL of each microorganism, adjusted to 0.5 McFarland standard (approximately 1.5 × 10^8 CFU/mL), was added to each well, except for the first column, which was used as a positive control, containing only BHI broth and microorganisms. The plate was incubated at 37 °C for 24 h and then read on a 600 nm spectrophotometer (BioTek, Winooski, VT, USA). The assay was repeated three times, and the results were expressed as the percentage of microorganism viability (V%) using the following formula:

Where At is the absorbance value of the tested wells, Ac is the absorbance value of the positive control wells, and Ab is the absorbance value of the negative control wells. The MIC was defined as the lowest concentration of the compound that inhibited 90% or more of the microorganism growth.

Determination of minimum bactericidal concentration (MBC)

Microorganisms were cultured in Brain Heart Infusion (BHI) broth for 24 h. Following this incubation period, a microbial stock was prepared, with a concentration ranging from 10⁵ to 10⁶ Colony Forming Units (CFU) per milliliter for each microorganism.

The Minimum Bactericidal Concentrations (MBCs) were determined by culturing wells exhibiting no bacterial growth on nutrient agar plates. These plates were subsequently incubated at a temperature of 37 °C for an overnight period.

The MBC was defined as the lowest concentration of the antimicrobial agent, resulting in the growth of fewer than four visible colonies. All experiments were performed in triplicate to ensure the accuracy and reproducibility of the results.

In vitro anti-proliferative assay

The cytotoxicity of C. colocynthis on the MCF-7 cell line was evaluated using the standard MTT colorimetric assay. A range of ten different concentrations of C. colocynthis, from 1 to 500 µg/mL, were prepared for this study.

MCF-7 cells were suspended in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and approximately 1% penicillin-streptomycin. Approximately 10⁴ MCF-7 cells were seeded in each microplate well to reach a 75–90% confluency. The microplates were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air.

On the following day, the medium in each well was replaced with 100 µL of C. colocynthis, previously prepared in RPMI medium. The plates were then incubated under the same conditions as the previous day. Subsequently, all wells were washed with Phosphate Buffered Saline (PBS) for approximately three minutes.

Following the washing step, 25 µL of MTT solution (4 mg/mL in medium) [3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well and the plates were incubated for an additional 4 h. The enzymatic reduction of MTT in living and metabolically active cells was the primary criterion for this assay, resulting in the formation of purple formazan crystals.

After removing the MTT solution, 100 µL of Dimethyl Sulfoxide (DMSO) was added, and the plates were incubated for 10 min to dissolve the formazan crystals. The plates were then shaken in an orbital manner for 5 min to ensure complete dissolution of the crystals.

The optical absorption of the solution was measured using an ELISA reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 540 nm. The experiments at this stage were performed in triplicate.

In this study, wells containing untreated MCF-7 cells served as the positive control (indicating 100% survival), and wells containing only culture medium served as the negative control (indicating 0% survival). Cell viability was calculated using the following equation:

%cell viability=[OD(cell) − OD(DMEM)][OD(cell + compound) − OD(compound)]​×100.

Statistical analysis

The statistical analysis was performed using the Microsoft Excel 2013 software. To investigate the differences in the mean viability percentage of the nanoparticles under test, One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc tests were employed for both antibacterial and cytotoxicity assessments. The experiments were replicated three times to ensure the reliability of the results. A p-value threshold of 0.05 was set to determine the statistical significance of the findings.

Results and discussions

Characterization

The nanoparticles were successfully synthesized and modified through a green protocol, as evidenced by Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-ray Analysis (EDAX), mapping, and Field Emission Scanning Electron Microscopy (FESEM) analyses. Figure 1 presents the FTIR spectra of 8HQ@ION and 8HQ@CCE-ION. The Fe-O tensile vibration mode, as indicated by the peaks in the 530–630 cm-1 region, validates the successful synthesis of these magnetic nanoparticles. Specifically, a broad and sharp peak at 574.01 cm-1 was observed for 8HQ@ION and 8HQ@CCE-ION, primarily associated with ION (18). Notably, this peak was more pronounced in the 8HQ@CCE-ION than in 8HQ@ION, suggesting a higher concentration of ION and, thus, a greater degree of magnetization in the 8HQ@CCE-ION. Furthermore, the spectra of ION and C, colocynthis exhibited peaks at wavelengths of 497.05, 821.28, 1106.95, and 3653.65 cm-1, corresponding to the sp2 alkene C-H band (disubstituted-E), in-plane C-H stretching vibration, FeOO-, and hydroxyl (-OH) functional groups, respectively.

Fig. 1.

FT-IR spectra of 8HQ@CCE-ION (green) and 8HQ@ION (black), with y-axis in transmittance (%T).

The 8HQ peaks exhibited significantly higher intensities at wavenumbers of 708.45, 1201.22, 1496.88, 1578.30, and 3023.76 cm-1, corresponding to CH bending, CO stretching, NO stretching, C = C stretching, and OH functional groups, respectively. The increased peak intensities at 1572.58 and 3639.37 cm-1 suggested a higher degree of oxidation and magnetization than the 8HQ@CCE-ION. Generally, the peaks at wavenumbers of 488.48, 551.33, 821.28, 1106.95, 1322.63, 1465.46, 2915.21, and 3639.37 cm-1 were associated with the alkene C-H band, halo compound C-Cl stretching, 1,4-disubstituted C-H bending, aliphatic ether C-O, aromatic ester C-O, alkane C-H bending, alkane C-H stretching, and alcohol O-H stretching, respectively. The peaks primarily resulted from the bending vibrations during the green synthesis process. The presence of these specific peaks indicated the successful formation of the 8HQ@CCE-ION nanocomposite through the green synthesis method. The increased levels of oxidation and magnetization in the peaks at 1572.58 and 3639.37 cm-1 hinted at potential enhancements in the properties of the nanocomposite compared to its original form³³. These findings confirmed the presence of Nanoparticle Surface (NPS) as the surface layer.

The nanoparticles synthesized via the green protocol were analyzed using Field Emission Scanning Electron Microscopy (FESEM), mapping, and Energy-Dispersive X-ray Spectroscopy (EDAX). These techniques confirmed the successful synthesis and transformation of the nanoparticles. The FE-SEM results showed that the shape of the nanoparticles (triangular, spherical, and hexagonal) changed based on the chemical composition, pH of the media, and concentration of the plant extract. Figure 2 presents the FE-SEM images of 8HQ@CCE-ION and 8HQ@ION. As shown in Fig. 3, the diameter of the ION nanoparticles ranged from 35 to 40 nm, with all particles displaying a spherical crystal and hexagonal structure. The mapping results demonstrated that elements were evenly spread throughout the nanoparticles, suggesting a consistent composition. In this regard, the in-depth study using FESEM, mapping, and EDAX revealed important information about the structure and properties of the synthesized nanoparticles. This laid the groundwork for further research into how they could be used in various fields.

Fig. 2.

FESEM images of (a, b) ION, (c, d) 8HQ, (e, f) 8HQ@ION, and (g, h) 8HQ@CCE-ION.

Fig. 3.

Analysis of XRD for ION, CCE-ION, 8HQ@ION and 8HQ@CCE-ION.

The X-ray diffraction (XRD) and EDAX patterns of ION, CCE-ION, 8HQ@ION, and 8HQ@CCE-ION are shown in Figs. 3 and 4a-d. EDAX spectra (Fig. 4) confirm the presence of characteristic elements in each variant: ION primarily shows Fe and O peaks, while 8HQ exhibits dominant C and O signals. Green synthesis with CCE introduces organic components (C, N, S, Cl) alongside Fe and O in CCE-ION and 8HQ@CCE-ION, with notably prominent Fe peaks in the green variants (Figs. 4b, d) compared to chemical counterparts (Fig. 4a, c). These qualitative differences indicate successful CCE integration, enhancing the nanocomposite’s organic-inorganic balance and supporting greater magnetite concentration in green-synthesized particles, which correlates with improved magnetic properties and bioactivity observed in antimicrobial assays.

Fig. 4.

EDAX spectra for (a) ION, (b) CCE-ION, 8HQ@ION, and (d) 8HQ@CCE-ION, showing characteristic elemental peaks. Green-synthesized variants (b, d) exhibit enhanced Fe signals and organic element incorporation compared to chemical counterparts (a, c), confirming successful CCE functionalization.

The EDAX mapping of 8HQ@CCE-ION (Fig. 5) reveals uniform spatial distribution of CCE-derived organic elements (C, O) alongside iron oxide throughout the nanocomposite structure. This even dispersion confirms successful green functionalization and strong phytochemical-nanoparticle interactions, validating the stability and homogeneity of the 8HQ@CCE-ION. The prominent iron signal distribution aligns with the enhanced Fe peaks observed in EDAX spectra (Fig. 4d), supporting the formation of a robust magnetite core suitable for magnetic separation and biomedical applications.

Fig. 5.

EDAX elemental mapping of 8HQ@CCE-ION, illustrating uniform distribution of CCE-derived carbon/oxygen (organic components) and iron oxide throughout the nanocomposite, confirming structural homogeneity and green synthesis efficacy.

Colloidal characterization and stability

The hydrodynamic diameter, polydispersity index (PDI), and zeta potential (ZP) of the synthesized nanostructures were assessed in phosphate-buffered saline (PBS, pH 7.4) to simulate physiological conditions. The results are summarized in Table 1. The corresponding particle size distribution graphs, obtained from dynamic light scattering (DLS), are presented in Fig. 6.

Table 1.

The hydrodynamic size, polydispersity index (PDI), ZP, and EE of ION, 8HQ@ION, CCE-ION, and 8HQ@CCE-ION in PBS at 4℃ for 24 h (pH 7.4). The data are mean ± SD (n = 3).

Nanostructure	hydrodynamic size (nm)	PDI	ZP (mV)
ION	60.6 ± 14.2	8.11 ± 4.04	+ 8.3 ± 1.5
8HQ@ION	79.0 ± 9.2	4.43 ± 1.09	-27.6 ± 2.9
CCE-ION	246.9 ± 17.7	0.08 ± 0.01	-34.2 ± 4.0
8HQ@CCE-ION	176.8 ± 13.1	7.07 ± 1.05	-44.5 ± 3.1

Note: Data is presented as mean ± SD.

*N is present as not related

Fig. 6.

Hydrodynamic size distribution by intensity. Dynamic light scattering (DLS) profiles for (a) ION, (b) 8HQ@ION, (c) CCE-ION, and (d) 8HQ@CCE-ION nanoparticles dispersed in PBS (pH 7.4). The sharp, monomodal peak in (c) for CCE-ION confirms its high monodispersity (PDI = 0.08), in contrast to the broad and multimodal distributions observed for the other formulations, which is consistent with their high PDI values.

The bare IONs exhibited a hydrodynamic size of 60.6 ± 14.2 nm. However, the notably high PDI value of 8.11 ± 4.04 indicates a highly heterogeneous and aggregated system. Consistent with this observation, the zeta potential was measured at + 8.3 ± 1.5 mV, which is critically low for achieving effective electrostatic stabilization.

Surface functionalization significantly altered the properties of the nanoparticles. Coating the iron oxide nanoparticles with 8-hydroxyquinoline (8HQ@ION) increased the hydrodynamic size to 79.0 ± 9.2 nm and shifted the zeta potential to a strongly negative value of -27.6 ± 2.9 mV, confirming successful functionalization. The PDI remained elevated at 4.43 ± 1.09, indicating a broad size distribution.

The green-synthesized nanoparticles (CCE-ION) displayed a larger hydrodynamic diameter of 246.9 ± 17.7 nm, attributed to the thick layer of biomolecules derived from Citrullus colocynthis extract. Notably, this sample exhibited an exceptionally low PDI of 0.08 ± 0.01, indicating a monodisperse, homogeneous population. The zeta potential measured − 34.2 ± 4.0 mV, reflecting excellent electrostatic stability.

In the case of the dual-coated system, 8HQ@CCE-ION, the hydrodynamic size was intermediate at 176.8 ± 13.1 nm; however, it exhibited a high PDI of 7.07 ± 1.05. Most remarkably, it possessed the most negative zeta potential among all formulations, measuring − 44.5 ± 3.1 mV.

The evaluation of colloidal stability is crucial for the biomedical use of nanoparticles, as it influences their interactions within biological environments and their stability over time³⁴. Our DLS and zeta potential measurements highlight the significant effects of different surface chemistries on iron oxide nanoparticles (IONs).

Magnetic properties and antimicrobial effect of amino and Lipoamino acid coated iron oxide nanoparticles

The bare IONs exhibit a near-neutral zeta potential of + 8.3 mV, along with a significantly high polydispersity index (PDI), indicating an unstable colloidal system. At a physiological pH of 7.4, which closely resembles the isoelectric point of magnetite (around pH 6–7), the electrostatic repulsion decreases, leading to rapid aggregation driven by van der Waals forces. This observation underscores the critical importance of surface modification for practical applications³³.

After coating with 8-hydroxyquinoline (8HQ), we observed a shift in zeta potential to -27.6 mV. This change is attributed to the coordination of 8HQ to the iron oxide surface. The deprotonation of the phenolic -OH group at physiological pH imparts a significant negative charge to the surface, enhancing electrostatic repulsion between nanoparticles. This zeta potential value exceeds the |±25| mV threshold typically associated with acceptable electrostatic stability³³. However, the persistently high PDI suggests that the 8HQ coating may not provide a perfectly uniform steric barrier, raising concerns about potential aggregation.

In contrast, the CCE-ION nanoparticles exhibited exceptional colloidal stability. The highly negative zeta potential of -34.2 mV confirms effective capping of the ION surface with anionic biomolecules derived from C. colocynthis extract, including polyphenols and proteins that contribute carboxylate and phenolate functionalities³⁵. The remarkably low PDI of 0.08 indicates the formation of a monodisperse population. This suggests that the phytochemicals in CCE function as both reducing and capping agents, as well as stabilizers that inhibit excessive growth and aggregation during synthesis, resulting in a homogeneous product. Combinations of high charge density and low PDI are commonly observed in green-synthesized nanoparticles using polyphenol-rich extracts³⁶.

The 8HQ@CCE-ION formulation exhibited the most negative surface potential, at -44.5 mV. This exceptionally negative zeta potential is likely due to a synergistic effect: the CCE establishes a biocompatible foundational layer, while the added 8HQ molecules introduce additional negative charges, resulting in a dense, highly charged corona. This finding aligns with research on dual-coated nanoparticles, which show that integrating diverse ligands enhances colloidal stability³⁷. Despite having a smaller hydrodynamic size than CCE-ION, the high PDI suggests a more complex, possibly heterogeneous coating morphology. This complexity may arise from competitive interactions between 8HQ and CCE phytochemicals or from the development of a less uniform coating layer. Nevertheless, the substantial negative zeta potential indicates robust long-term electrostatic stability, which would effectively counteract aggregation in ionic media such as phosphate-buffered saline (PBS).

Morphology and core size analysis by transmission electron microscopy

The morphology and core diameter of the synthesized nanoparticles were analyzed using Transmission Electron Microscopy (TEM). Figure 7 displays representative TEM micrographs along with the corresponding core size distributions, which were obtained by manually sizing over 100 particles per sample. The bare iron oxide nanoparticles (IONs) (a) showed a quasi-spherical morphology with an average core diameter of 23 ± 2.4 nm. After functionalization with 8-hydroxyquinoline (b), the particles transformed into 8HQ@ION particles, exhibiting an average core size of 41 ± 6.5 nm, indicating the formation of a distinct organic layer surrounding the inorganic core. The green-synthesized CCE-ION nanoparticles (c) demonstrated a significant increase in core size, measuring 89 ± 14.9 nm, and were encased in a thick, electron-lucent coating characteristic of the biomolecular corona derived from the C. colocynthis extract. Finally, the dual-coated 8HQ@CCE-ION nanoparticles (d) displayed a core size of 66 ± 9.0 nm, with a visible coating that appeared denser than that of the bare IONs, yet structurally distinct from the coating of the CCE-ION nanoparticles alone.

Fig. 7.

Morphology and core size analysis of synthesized nanoparticles. Representative Transmission Electron Microscopy (TEM) images for (a) ION, (b) 8HQ@ION, (c) CCE-ION, and (d) 8HQ@CCE-ION. The mean core diameter ± standard deviation was determined by manually measuring over 100 individual particles from multiple images. Scale bars represent 100 nm.

The detailed characterization of our nanoparticles using TEM, DLS, and zeta potential measurements provides important insights into their physical and colloidal properties, which are critical for their effectiveness in pharmaceutical applications.

A key comparison between the TEM-determined core size and the DLS-measured hydrodynamic diameter shows that, across all samples, the hydrodynamic diameter is significantly larger than the TEM core size. This well-documented phenomenon occurs because DLS measures the effective diameter of the particle core, including its solvation shell and any surface modifications, in a hydrated state, while TEM focuses solely on the dry, inorganic core in a high-vacuum environment²⁷. The degree of this difference varies significantly depending on the nature of the surface coating.

For bare iron oxide nanoparticles (IONs), we observe a moderate increase from a TEM-determined size of 23 nm to a DLS-measured hydrodynamic size of 60.6 nm. This difference, compounded by a very high polydispersity index (PDI) and a near-neutral zeta potential, visually indicates aggregation. The uncoated particles are prone to forming larger clusters in solution, leading to a higher observed hydrodynamic diameter³⁴.

In the case of 8HQ@ION, the core size determined by TEM increased to 41 nm, confirming the successful formation of a coating from 8HQ on the particle surface. The corresponding hydrodynamic size of 79.0 nm supports this observation, with a difference of approximately 38 nm attributed to the hydrated layer of 8HQ and the ionic double layer surrounding the particles. The shift to a strong negative zeta potential of -27.6 mV confirms the practical introduction of charged phenolate groups. However, the elevated PDI in both TEM and DLS suggests that the coating process may lead to variability in coating thickness or minor aggregation, a challenge noted in other studies of small-molecular coatings³⁶.

The most remarkable findings stem from the green-synthesized CCE-ION. TEM analysis reveals a significantly larger core of 89 nm, accompanied by a prominent biomolecular corona. The hydrodynamic diameter of 246.9 nm indicates the formation of an extensive, hydrated polymer brush composed of CCE phytochemicals, including polysaccharides and proteins. This is typical for green-synthesized nanoparticles, where plant extracts serve as both in situ capping and stabilizing agents, resulting in a substantial organic shell³⁵. The combination of a robust steric barrier, a high negative zeta potential of -34.2 mV, and an exceptionally low PDI of 0.08 indicates that the CCE effectively stabilizes the nanoparticles, making them both electrostatically and sterically stabilized as well as remarkably monodisperse. This aligns with recent literature emphasizing the benefits of plant extracts in producing high-quality, stable nanomaterials^26,34,35,38.

The dual-coated 8HQ@CCE-ION offers a compelling case. Its core size, as determined by TEM, is 66 nm, which is noticeably smaller than that of CCE-ION (89 nm). This suggests that incorporating 8HQ during green synthesis can influence the nucleation and growth of the iron oxide core, leading to smaller, more uniform inorganic particles. Despite its smaller core, the hydrodynamic size of 176.8 nm is significant, accompanied by the most negative zeta potential measured at -44.5 mV. This indicates a thick final coating with a substantial charge density, likely resulting from the synergistic coordination of 8HQ on the CCE-coated surface, which introduces additional anionic sites. The elevated PDI may reflect complexities arising from the dual-coating process, leading to a particle population with variable coating densities. This innovative strategy of combining green and chemical coatings represents a promising approach for engineering surfaces with tailored physicochemical properties³⁷.

Antibacterial and antifungal performance

Minimum inhibitory concentration (MIC)

Antibacterial activity results

The antibacterial activity of nanoparticles was tested against four species of human pathogenic bacteria: E. coli, S. aureus, E. faecalis, and P. aeruginosa. Results indicated that CCE-ION effectively inhibited the growth of all test organisms. The order of sensitivity, based on the width of the inhibition zone and absorption at 600 nm (Table 2; Fig. 8), was as follows: P. aeruginosa > E. coli > S. aureus > E. faecalis.

Table 2.

MICs and MBC / MFCs Values of compounds against microorganisms.

Microorganisms		CCE-ION		CCE
		MIC(µg/mL)	MBC/MFC(µg/mL)	MIC(µg/mL)	MBC/MFC(µg/mL)
Gram positive	Staphylococcus aureus	500	500	250	500
	Enterococcus faecalis	1000	1000	500	500
Gram negative	Escherichia coli	250	250	125	250
	Pseudomonas aeruginosa	125	250	62.5	125
Yeast	Candida	500	500	250	250

Fig. 8.

Effects of different concentrations of tested nanoparticles on the viability of various microorganisms. In three independent tests, the mean ± SD (standard deviation) is indicated by each.

Antifungal activity results

The antifungal activity of nanoparticles was assessed against the fungus C. albicans. CCE-ION effectively inhibited growth, with sensitivity ranked below S. aureus but above E. faecalis in the overall order (Table 2; Fig. 8).

Comparative evaluation with plant extract

The comparison was carried out for the antibacterial activity of C. colocynthis/Fe and with that of C. colocynthis, a plant extract reported to possess antimicrobial properties. C. colocynthis/Fe nanoparticle showed a higher antibacterial effect than C. colocynthis’s essential oil (EO) on S. aureus, E. faecalis, E. coli, and P. aeruginosa at concentrations ranging from 62.5 to 250 µg/mL (Fig. 8). In this case, C. colocynthis/Fe nanoparticles showed a more potent antibacterial effect on P. aeruginosa at 125 µg/mL, followed by S. aureus at 250 µg/mL. In contrast, neither the essential oil (EO) nor the EO/Fe nanoparticles showed any antibacterial activity against E. faecalis at concentrations below 500 µg/mL. A higher antifungal effect was observed with C. colocynthis/Fe than with C. colocynthis on C. albicans at 125 µg/mL concentrations. These findings indicate that creating iron oxide nanoparticles through the green synthesis method using C. colocynthis extract may decrease the presence or effectiveness of the phytochemicals like flavonoids, alkaloids, and terpenoids responsible for the antimicrobial properties^20,21.

In mechanistic point of view, iron oxide nanoparticles can interact with the bacterial cell wall or membrane. This interaction may change the permeability or integrity of the cell, affecting how the C. colocynthis extract enters the bacterial cells^22,23. Additionally, the iron oxide nanoparticles might create oxidative stress or reactive oxygen species (ROS) in the bacterial cells. This stress could interfere with the antimicrobial action of the C. colocynthis extract or lead to bacterial resistance or adaptation^21,24.

It is suggested that the improved antibacterial activity of C. colocynthis/Fe is attributed to the smaller size and larger surface area of the nanoparticles. This allows them to better penetrate bacterial cell membranes, leading to a stronger bactericidal effect. It is believed that iron ions from C. colocynthis/Fe attach to the negatively charged bacterial cell wall, leading to its disruption. This results in protein denaturation and, ultimately, cell death. Additionally, the antifungal effect of C. colocynthis/Fe might include direct interaction between nanoparticles and the surfaces of fungal cells. This interaction alters membrane permeability, enabling nanoparticles to induce oxidative stress in fungal cells, leading to inhibition of cell growth and cell death. These mechanisms are consistent with previous reports of membrane damage and oxidative stress caused by metal oxide nanoparticles in microbial cells^19,21,22,39.

Our study demonstrates that C. colocynthis/Fe has potent antimicrobial activity against human pathogenic microorganisms. However, the optimal concentration and the interaction with C. colocynthis extract may vary depending on the type of microorganism. Further studies are needed to elucidate the exact mechanism of action and the optimal concentration of C. colocynthis/Fe for antimicrobial applications. Our findings also indicate that C. colocynthis/Fe may have potential as an antimicrobial drug in medicine, as it has a higher antimicrobial effect than other plant-derived nanoparticles^26,31,40.

Assay of in vitro cell toxicity

In the MTT assays, untreated cells served as positive controls (100% viability), while wells with only culture medium acted as negative controls (0% viability); DMSO was used solely for formazan crystal dissolution post-assay and not as a vehicle control, as compounds were prepared directly in RPMI medium. Blank FeNPs (without CCE) were not tested for cytotoxicity in this study, as our focus was on evaluating the synergistic effects of the 8HQ@CCE-ION nanocomposite. We evaluated the cytotoxicity of C. colocynthis extract and FeNPs synthesized by 8-hydroxyquinoline Fe₃O₄ using C. colocynthis on two human cancer cell lines: MCF-7 (breast) and Hep-G2 (liver) using the MTT assay (Fig. 9). To our knowledge, no previous studies have reported the anticancer activity of these compounds on these cell lines. Different concentrations (1, 10, 50, 100, 200, and 500 µg/mL) of C. colocynthis extract and C. colocynthis-FeNPs were tested and compared with the control group (Table 3). We found that both C. colocynthis extract and C. colocynthis-FeNPs had high cytotoxic effects at high concentrations (200 and 500 µg/mL) on both cell lines, with moderate activity levels according to the US National Cancer Institute’s Nanotechnology (Nanotechnology Characterization Laboratory)²⁸. However, the cytotoxic effect of C. colocynthis extract decreased significantly at concentrations below 200 µg/mL. In comparison, the cytotoxic effect of C. colocynthis-FeNPs remained at lower concentrations (50 and 100 µg/mL) for MCF-7 and HepG2 cells, respectively.

Fig. 9.

Effect of tested concentrations of all compounds on the viability of MTT assay on MCf-7 and Hep-G2 cells after 24 campers with control.

Table 3.

Key summary of cytotoxicity data for CCE and 8HQ@CCE-ION against MCF-7 and Hep-G2 cancer cell lines, based on MTT assay after 24 h exposure.

Compound	Cell Line	IC₅₀ (µg/mL)	% Viability at 100 µg/mL	% Viability at 200 µg/mL	% Viability at 500 µg/mL
CCE	MCF-7	˃500	79.30 ± 1.19	65.60 ± 0.99	53.89 ± 5.35
CCE	Hep-G2	223	61.29 ± 0.21	50.70 ± 3.26	41.64 ± 2.56
8HQ@CCE	MCF-7	489	78.16 ± 1.69	62.01 ± 2.50	49.53 ± 2.45
8HQ@CCE	Hep-G2	183	60.40 ± 1.84	47.92 ± 2.72	38.28 ± 2.67

Differences in viability between 8HQ@CCE-ION and CCE were statistically significant at concentrations ≥ 100 µg/mL.

Cancer is a major global health problem that causes millions of deaths every year [72, 73]. Adverse effects and high costs of conventional therapies have driven the exploration for alternative drugs with reduced side effects and costs. Plants and herbal extracts have been widely used as sources of natural compounds with anticancer properties [74, 75]. C. colocynthis is a plant that has been reported to have antimicrobial and antioxidant activities due to the presence of phenolic acids and flavonoid compounds such as thymol, carvacrol, ρ-cymene, and γ-terpinene^31,32. However, the anticancer mechanism of C. colocynthis extract is not well understood. Some studies have suggested it may involve the delay or inhibition of oxidative damage caused by free radical and non-free radical species³².

FeNPs are a type of nanomaterials that have attracted attention for their potential applications in biomedicine, especially in cancer therapy^27,29,33. The exact anticancer mechanism of FeNPs is also unclear. Still, some hypotheses have been proposed, such as the induction of apoptosis and cell cycle arrest through the mitochondrial pathway³⁰ or the direct contact and cytotoxicity of FeNPs with the cell surfaces due to their small size and shape³³. In this regard, the enhanced cytotoxicity of 8HQ@CCE-ION compared to free CCE (Table 3) may involve ROS-mediated apoptosis, where iron oxide nanoparticles generate reactive oxygen species that induce oxidative stress and programmed cell death in cancer cells^27,30. Additional plausible mechanisms include mitochondrial dysfunction, leading to impaired energy production and apoptosis, and cell cycle arrest at G0/G1 or G2/M phases, disrupting proliferation in a way that in consistent with reports on iron-based nanoparticles in Hep-G2 and MCF-7 lines^27,30. These mechanisms propose that 8HQ@CCE-ION could be an effective treatment for cancer by targeting and destroying cancer cells. Additionally, studies have shown that FeNPs have low toxicity towards normal cells, making them a promising candidate for targeted cancer therapy. Further research is needed to fully understand the mechanisms of action of FeNPs in cancer treatment and to optimize their use in clinical settings.

Conclusions

This research presents a novel, eco-friendly method for synthesizing 8-hydroxyquinoline-functionalized iron oxide nanoparticles using Citrullus colocynthis extract. The resulting 8HQ@CCE-ION nanocomposite demonstrated a dual-action capability, exhibiting both significant antimicrobial and anticancer potential. The green synthesis approach offers a sustainable alternative to traditional chemical methods, while the functionalization with 8HQ enhances the therapeutic efficacy of the nanoparticles. Specifically, the nanocomposite showed strong antimicrobial activity against clinically relevant pathogens, including P. aeruginosa, E. coli, S. aureus, E. faecalis, and C. albicans, and significant cytotoxicity against MCF7 and Hep-G2 cancer cells. These results highlight the potential of this nanoplatform to address the growing challenges of drug-resistant infections and cancer. Future studies should focus on optimizing the synthesis process, investigating the precise mechanisms of action, and evaluating the in vivo therapeutic potential of the 8HQ@CCE-ION nanocomposite.

Author contributions

Ahmad Gholami and Milad Mohkam performed research and laboratory work. Khadije Yousefi, Mohammad Hashem Hashempur and Kamran Bagheri Lankarani performed research and analyze data and manuscript reviewer. Navid Omidifar and Seyyed Mojtaba Mousavi planning and supervising the study and statistical analysis and wrote the manuscript. Wei-Hung Chiang and Chin Wei Lai managed the laboratory work, including sample collection and processing as well as contributed to writing sections of the manuscript and ensured compliance with ethical standards.

Funding

This research is funding by Shiraz University of Medical Sciences, Shiraz, Iran.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.