Document Type : Original Research Article
Subjects
Introduction
Laser ablation in liquid (PLAL) is one of the emerging modern environmentally-friendly methods of producing high-purity nanoparticles without any chemical precursors and surfactants [1]. The method is physical and top-down whereby high-intensity pulsed laser beams are directed to a submerged liquid target of solid iron, resulting in rapid heating of surface, melting, and vaporization, which is followed by forming of a plasma plume and ultrafast quenching to produce iron oxide nanostructures [2, 3].
The targets that are normally utilized in this process are pure iron or iron-based alloys and Nd:YAG lasers with a wavelength of 1064 or 355 nm and pulse durations of femtosecond-nanosecond allow the fine tuning of particle formation [4]. The rate of ablation is modulated by laser parameters including pulse energy, wavelength, irradiation time, and the concentration of particles, colloidal stability, and agglomeration degree, and the liquid phase (e.g., water vs. ethanol) has a strong impact on oxidation state and phase composition [5-9].
Magnetic nanoparticles (MNPs) and especially IONPs have attracted much interest in the oncology field because of their magnetic properties, biocompatibility and ease of surface functionalization, which allows them to be used in targeted drug delivery, imaging and image-guided therapy [10, 11]. Compared to standard chemotherapy and radiotherapy, which can often be restricted by the systemic contribution of the drug and lack of tumor specificity, MNP-based systems may be utilized in magnetic targeting to tumors, magnetic hyperthermia [12, 13], and ferroptosis-/ROS-mediated programmed cell death, as well as used as contrast agents in magnetic resonance imaging (MRI) and in tumor real-time monitoring [14, 15].
Among the many available synthesis methods, laser ablation in liquid is especially appealing to synthesize iron oxide nanoparticles to be used in biomedical applications due to its simplicity, comprising no templates, no reagents, minimal contamination, and control of particle size, morphology, crystallinity, and magnetic qualities by simply changing laser fluence and exposure conditions. PLAL has high purity, small size distribution, and high reproducibility when compared to the chemical reduction/precipitation techniques, essential to clinical translation [2, 3, 16, 17].
Iron oxide nanoparticles are also the best to be used in laser mediated cancer therapy as they can take up light and convert this into heat and can be used to achieve localized photo-thermal ablation of tumor tissue when exposed to specific wavelengths [18-20]. These nanoparticles can also undergo photodynamic processes under certain conditions, wherein they produce reactive oxygen species (ROS) that induces oxidative stress, mitochondrial dysfunction, and apoptosis, thus, merging photothermal and photodynamic effects on a single platform [20, 21].
In addition, the superparamagnetic characteristics of iron oxide nanoparticles enable them to be accumulated in the tumor sites using external magnetic fields and to be actively targeted when conjugated with tumor-specific ligands or the antibodies [22-24]. The multimodal nanotheranostic approach of magnetic guidance, photothermal heating, ROS-based photodynamic action is expected to provide high level of efficacy, low level of invasiveness and low level of off-target toxicity in the treatment of advanced cancer therapy.
This work aims to synthesize IONPs via pulsed laser ablation in liquid using varying energies (300, 400, and 500 mJ) on an iron target in double-distilled water, characterize their structural, morphological, magnetic, and optical properties using XRD, FESEM, VSM, and UV-Vis spectroscopy, and evaluate their selective anticancer efficacy against HepG2 cancer cells versus RD normal cells through crystal violet cytotoxicity assays, with and without 532 nm laser irradiation to demonstrate combined photothermal and photodynamic effects for targeted tumor therapy.
IONPs were synthesized via PLAL using a Q-switched Nd:YAG laser (λ = 1064 nm, pulse width = 10 ns, repetition rate = 1 Hz). A high-purity iron target (99%, 2 × 2 cm pellet, Sigma -Aldrich) was placed at the bottom of a glass vessel containing 3 mL double-distilled water (DDW) under constant magnetic stirring, and irradiated at laser energies of 300, 400, and 500 mJ per pulse for a fixed duration. A laser beam was focused on the target surface causing rapid warming, melting, and vaporization to generate a plasma plume, which expanded into the liquid medium, and then ultrafast quenching and interaction with dissolved oxygen facilitated the nucleation, growth and oxidation of iron species to IONPs, which consisted in a change of color to dark red colloid. Laser fluence was used to tune the particle size, concentration, and phase composition, higher energies gave rise to higher ablation rates and smaller and more crystalline nanoparticles.
The phase composition, crystallinity, and size of the crystallites of the (IONPs) were determined through the X-ray diffraction (XRD; Shimadzu XRD-6000) analysis using Cu Kα radiation (λ = 1.5406 Å) at 2θ range of 20°–80° with a step size of 0.02 °and a scan rate of 5°/min. Phase identification was done against standard JCPDS databases through diffraction patterns.
Field emission scanning electron microscopy (FESEM; FEI NOVA NanoSEM450) was employed to examine nanoparticle morphology, shape, and size distribution.
Magnetic properties were evaluated using a vibrating sample magnetometer (VSM) at room temperature (300 K) over a field range of ±15 kOe, revealing size-, crystallinity-, and defect-dependent superparamagnetic behavior.
HepG2 (human hepatocellular carcinoma) cancer cells and RD (normal rhabdomyosarcoma) cells were independently cultured in 30 cm² flasks using RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified 5% CO₂ atmosphere for 24 h to reach 80–90% confluency prior to experiments.
Cells were harvested from 25 cm² flasks by trypsin-EDTA detachment, neutralized with 20 mL serum-supplemented RPMI-1640, and resuspended. Aliquots (0.2 mL, ~10⁴ cells/well) were seeded into 96-well flat-bottom plates in triplicate and incubated at 37°C, 5% CO₂ for 24 h to allow adherence. Medium was replaced with fresh medium containing IONPs at concentrations of 6.25, 12.5, 25, 50, and 100 μg/mL (three replicates per concentration), with untreated cell controls. After 24 h exposure ± 532 nm laser irradiation (5 min), viability was assessed by crystal violet assay: cells were fixed, stained (100 μL/well, 20 min), washed, destained, and absorbance measured at 492 nm using a microplate reader. Cytotoxicity (% inhibition) was calculated as:
Inhibition rate = (A – B) /A × 100 (1)
where A = mean OD of control wells and B = mean OD of IONP-treated wells.
Irradiation cancer cell by laser Diode
IONP-treated cells were irradiated using a 532 nm diode laser (5 min, tuned to nanoparticle absorption band) to activate photothermal and photodynamic effects. The laser induces: (i) photothermal heating via light absorption and conversion to localized hyperthermia (42–60°C), denaturing proteins, disrupting membranes/DNA, and triggering apoptosis/necrosis; (ii) photodynamic ROS generation (- OH, O₂⁻, ¹O₂) through photoexcited electron transfer interacting with water/oxygen, causing oxidative stress, lipid peroxidation, mitochondrial dysfunction, and caspase-mediated cell death. Complementary mechanisms include magnetic guidance for tumor targeting, potential functionalization with antibodies/peptides/folic acid, vascular disruption, and immune activation via antigen release.
The statistical analysis of the results was conducted by using Graph Pad Prism Version 6 analysis software and analysis of variance. Means were compared by using the Duncan multiplex experiment, wherein significant differences were observed at a probability threshold of P < 0.05.
Figure 2 presents XRD patterns of IONPs synthesized at laser energies of 300, 400, and 500 mJ, with 2θ (Bragg angle) on the x-axis and diffracted X-ray intensity (arbitrary units, a.u.) on the y-axis. Diffraction peaks, indexed with Miller indices (hkl), were identified by matching against the JCPDS reference for hematite. Peak intensity and sharpness increased with laser energy, indicating enhanced crystallinity and reduced lattice strain at higher fluences due to greater atomic mobility during nucleation. Broader peaks at 300 mJ suggest smaller crystallite sizes (~43 nm) or higher defect density, while narrower peaks at 500 mJ reflect improved crystallization (~33 nm) and potential preferred orientation, as evidenced by relative intensity variations across (hkl) planes.
Crystallite sizes were calculated using the Debye-Scherrer equation:
D =
(2)
where DDD is the mean crystallite size, KKK = 0.94 (Scherrer constant), λ\lambdaλ = 1.5406 Å (Cu Kα wavelength), β\betaβ = peak full width at half maximum (FWHM, radians), and θ\thetaθ = Bragg angle. The most prominent peak yielded crystallite sizes of 43 nm (300 mJ), 40 nm (400 mJ), and 33 nm (500 mJ), confirming energy-dependent refinement of nanoparticle crystallinity.
Fig. 3 of the FESEM depicts irregular, agglomerated nanoparticles whereby the primary particles are mostly spherical with a few polyhedral ones forming clusters probably through van der Waals attractions and/or partial sintering.
At 500 mJ, the higher laser energy enhances ablation and plasma formation, increasing fragmentation and nucleation rates and thus favoring the generation of smaller nanoparticles while inhibiting secondary growth and aggregation through stronger repulsive interactions. In contrast, at 300–400 mJ, reduced ablation efficiency and lower thermal input lead to incomplete fragmentation, slower nucleation and growth, and consequently larger particles with a higher tendency to aggregate. Overall, the measured particle diameters range from about 46 to 142 nm, indicating a polydisperse system in which the smaller nanoparticles likely represent primary particles, whereas the larger ones are aggregates or agglomerates[7, 25].
Laser-induced heating can reduce the size of IONPs, and when the particle size falls below about 20 nm the system can exhibit superparamagnetic behavior characterized by a narrow hysteresis loop and reduced coercivity (Hc), as observed for the smaller particles produced at 500 and 400 mJ in Fig. 4. In addition, higher laser energies may introduce lattice defects, strain, or oxygen vacancies in the IONPs, which can modify their magnetic properties, often enhance the saturation magnetization (Ms) and alter Hc. Conversely, at 300 mJ the larger particles show higher coercivity and a wider hysteresis loop consistent with more stable ferromagnetic-like behavior, while a different defect structure at this lower energy may contribute to the observed decrease in Ms
The optical spectra of IONPs samples prepared by laser ablation at 300, 400, and 500 mJ (Fig. 5) show that the absorbance decreases with increasing wavelength, with noticeably higher absorption in the short-wavelength region, which can be attributed to electronic transitions from the valence band to the conduction band and is consistent with a nanostructured material. In addition, the sample produced at the highest laser energy exhibits the greatest overall absorbance, implying more efficient vaporization of the IONPs target, a higher nanoparticle concentration, and likely smaller or more densely packed nanoparticles that provide more light-absorbing active sites. This enhanced absorption can be associated with size-dependent effects such as surface plasmon resonance or quantum confinement in the nanoscale regime, which are known to increase the optical response of metal oxide nanoparticles.
Two cell lines, the hepatocellular carcinoma cell line HepG2 and the normal muscle cell line RD, were treated with IONPs at concentrations of 6.25, 12.5, 25, 50, and 100 μg/ml and irradiated with a 532 nm laser diode for 5 min, then incubated for 24 h at 37 °C. The viability data (Tables 1 and 2) show a clear concentration-dependent inhibitory effect of IONPs on both HepG2 and RD cells after 24 h, with the strongest cytotoxicity observed for nanoparticles synthesized at 500 mJ, which likely reflects their higher concentration in the colloid and smaller size, and thus larger specific surface area, compared with particles produced at 300 and 400 mJ. In line with previous reports [26-29], on laser-ablated iron oxide nanomaterials, the increased surface area and reactivity of smaller IONPs enhance their interaction with cells and consequently their inhibitory potential on cell proliferation. Furthermore, Fig. 6 illustrates that laser-activated IONPs can act as targeted therapeutic agents, where their magnetic and photothermal/photodynamic responses promote localized heating, reactive oxygen species generation, mitochondrial dysfunction, and ultimately apoptosis in tumor cells, thereby improving tumor damage while potentially limiting systemic toxicity, consistent with earlier studies [30-33] on IONP-mediated cancer therapy.
The synthesis of IONPs by laser ablation represents a green and efficient route for producing high-purity colloids without chemical precursors or stabilizing agents. XRD analysis in this work confirms the formation of iron oxide phases under the applied ablation conditions, while FESEM images show nearly spherical nanoparticles in the nanometer range whose size can be tuned by adjusting laser energy, pulse characteristics, and wavelength. The combination of favorable biocompatibility and magnetic behavior makes such IONPs attractive for biomedical use, including magnetic hyperthermia, targeted delivery, and MRI contrast enhancement, where superparamagnetic-like responses are particularly advantageous. The data in the cytotoxicity results show that the particles have the capacity to selectively induce apoptosis in cancer cells and relatively less effect on normal cells, which suggest their potential use as anticancer applications. Altogether, laser ablation is the clean and controllable method of preparing iron oxide nanoparticles to be used in cancer treatment, and further research needs to refine the size control and surface functionalization of particles and extensive in vivo tests to bring the potential of these nanoparticles to full realization, according to the current development trends in nanomedicine.
The authors utilized artificial intelligence tools, namely Perplexity.ai, to enhance the clarity and language quality of this manuscript throughout its preparation. All suggestions and content provided by the AI were thoroughly reviewed and revised by the authors, who take full responsibility for the accuracy and integrity of the final version.
The authors declare no conflict of interest.
