TEM analysis evaluated the size and morphology of NPs. Figure 2a and b shows images of α-Fe2O3NPs. In Fig. 2c, the corresponding histogram was plotted using TEM images of the nanoparticles and analyzed with Image J software. The average particle size was estimated to be 45 nm with a standard deviation of 11 nm. Besides, they have a somewhat spherical shape and some porosity on their surface.
a, b TEM images of α-Fe2O3 NPs, c particle size distribution from TEM images, analyzed using Image J (mean:45 nm; SD: 11 nm).
The XRD pattern of α-Fe2O3NPs is shown in Fig. 3a, which agrees with the standard pattern data (JCPDS card No. 33–0664)37. The peaks appearing in the 24.10, 33.09, 35.61, 40.82, 49.44, 54.00, 57.46, 62.41, and 63.95 degrees have been attributed to the diffraction planes (012), (104), (110), (113), (024), (116), (018), (214) and (300) of NPs crystalline respectively. All sharp and narrow peaks appearing in the range of 2θ indicate the crystalline nature and the high purity of α-Fe2O3NPs extracted by the combined of green synthesis and hydrothermal method. In addition, the XRD pattern of nanocomposite is shown in Fig. 3b. In this pattern, all the characteristic peaks related to α-Fe2O3NPs and the characteristic broad peak at the angle of 2θ = 20.26 related to CS biopolymer have been observed, confirming the successful formation of this nanocomposite38.
XRD pattern of a α-Fe2O3 NPs, b CS-α-Fe2O3 nanocomposite.
The crystallite size of α-Fe2O3NPs can be estimated by using the Williamson–Hall method (Eq. 1) 39:
$$beta cos theta =left( {0.89lambda } right)/d+4varepsilon sin theta$$
(1)
In this equation, β is full width at half maximum (FWHM), θ is the diffraction angle, λ is the X-ray wavelength, d is the average crystallite size, and ε is the lattice strain. The crystallite size of α-Fe2O3NPs was determined by drawing a diagram (Fig. 4) and calculating the width from the origin. With these interpretations, the crystallite size was estimated to be 43 nm.
Williamson–Hall diagram of –Fe2O3 crystallite.
One of the important characteristics of colloidal suspensions is the tendency of their particles to stick together. In an aqueous environment, particles continuously interact, and the stability of such solutions is determined based on these interactions. In order to create a stable solution, short-range repulsive forces are needed, among which we can mention the steric interfacial forces that play an important role in stabilizing suspensions. Therefore, these electric forces create layers around the NPs, and this issue plays a significant role in the value of zeta potential and ultimately prevents particles from agglomerating40. In this research, the stability of the CS-α-Fe2O3 nanocomposite solution originates from the strong bonding of CS on the surface and the charge of α-Fe2O3NPs, which causes repulsion between them. The CS, a biocompatible and biodegradable polymer, forms a protective layer around the α-Fe2O3NPs, preventing them from agglomerating and thus contributing to the stability of the solution. In order to study the stability of α-Fe2O3NPs and CS-α-Fe2O3 nanocomposite solutions with pH 5.11 and viscosity 0.9327 mPas, zeta potential analysis as a critical parameter was performed, and their results are shown separately in Fig. 5a and b. In these graphs, the distribution function of the zeta potential of individual NPs and nanocomposite in the solution is presented as a percentage, and the average zeta potential of unmodified α-Fe2O3NPs and CS-α-Fe2O3 nanocomposite was measured 29.93 and 36.67 mV, respectively. Also, by comparing the two graphs, the results indicate that adding CS to α-Fe2O3NPs narrows the peak width of the graph, which indicates that a large percentage of nanocomposites have the same zeta potential, and this issue was evaluated as stabilization of CS-α-Fe2O3 nanocomposite suspension. In an experiment, two solutions of NPs and CS-α-Fe2O3 nanocomposite with the same concentration of 5 mg/ml were prepared, and the role of adding CS in stabilizing the α-Fe2O3NPs solution after 72 h is shown in Fig. 5c. Another result obtained from the zeta potential of CS-α-Fe2O3 nanocomposite suspension indicates the net positive charge of these nanocomposites. Furthermore, these nanocomposites can stick and penetrate cancer cells with a negative charge through electrostatic forces41.
Zeta potential distribution function diagram of a α-Fe2O3 NPs, b CS-α-Fe2O3 nanocomposite, c comparing the stability of Fe2O3 NPs solution and CS-Fe2O3 nanocomposite solution after 72 h.
Figure 6a and b shows the UV–visible absorption spectrum of α-Fe2O3NPs and CS-α-Fe2O3 nanocomposite between 200 and 900 nm. The absorption peak for α-Fe2O3NPs and CS-α-Fe2O3 nanocomposite is 450 and 380 nm, respectively. This result is significant and reassuring, as it aligns with the data of another research36 enhancing our confidence in the findings. Considering the absorption in the infrared region, this issue guarantees the wide potential of this nanocomposite to be used in cancer treatment by PTT and PDT with an 808 nm laser.
UV–Visible absorption spectrum of a α-Fe2O3 NPs, b CS-α-Fe2O3 nanocomposite.
The optical band gap of α-Fe2O3 NPs can be calculated by using Tack’s equation (Eq. 2)42.
$${(alpha hnu )^n}=Aleft( {hnu – {E_g}} right)$$
(2)
In this equation, α is the absorption coefficient, A is a constant, hν is the photon energy, and n is a constant that depends on the nature of the electron transition, which is 2 for direct transition and 1/2 for indirect transition. Since α-Fe2O3 NPs have a direct energy gap, the graph of (αhν)^2 in terms of energy is drawn in Fig. 743. The value of the energy gap associated with α-Fe2O3 NPs was approximated at 1.8 eV using the extrapolation method, which is comparable to the result of another research44. Also, our Experimental investigation indicates that the NPs exhibit an indirect band gap of roughly 1.7 eV.
α-Fe2O3 NPs Tauc diagram.
FTIR analysis was performed in the 400–4000 cm− 1 wave number range to determine the bonds and functional groups on the surface of CS powder, α-Fe2O3NPs, and CS-α-Fe2O3 nanocomposite. The FTIR spectrum of α-Fe2O3NPs is shown in Fig. 8a. A broad peak in the range of 3445 cm− 1 is assigned to the stretching vibration between oxygen and hydrogen belonging to the functional groups of hydroxyl and water molecules, which proves the absorption of some water at the surface of α-Fe2O3NPs45. Also, two sharp absorption peaks in the range below 1000 Cm− 1 indicate the main characteristics of α-Fe2O3 NPs, which are attributed to the stretching frequencies of metallic iron. The high frequency peak in 526 cm− 1 refers to Fe-O deformation in tetrahedral and octahedral environments. At the same time, the peak at low frequency in the range of 450 cm− 1 refers to the Fe–O shape change in the octahedral environment of hematite46. In the spectrum of CS powder, the peaks appearing in the 3445 cm− 1 and 3360 cm− 1 range are related to O–H and N–H stretching vibrations, respectively, and intra molecular hydrogen bonds. The peaks around 2925 cm− 1 and 2854 cm− 1 are attributed to symmetric and asymmetric C–H stretching vibrations, which are common bonds characteristic of polysaccharides. The remaining presence of N-acetyl groups was confirmed by peaks in the range of 1741 cm− 1 related to C=O stretching vibrations. Also, C–H bending vibrations and the symmetric deformation were confirmed by the peak in 1369 cm− 1. The peak appearing in 1020 cm− 1 is attributed to C–O stretching vibrations. All peaks related to CS material are shown in Fig. 8b47. By analyzing the FTIR analysis of CS-α-Fe2O3 nanocomposite, all the characteristic peaks of the infrared Fourier transform of α-Fe2O3NPs except Fe–O and all the characteristic peaks of CS were observed (Fig. 8c).
FTIR spectrum of a α-Fe2O3 NPs, b CS, c CS-α-Fe2O3 nanocomposite.
Three different concentrations of CS-α-Fe2O3 nanocomposite solution were prepared to study PTT effects using an ultrasonic bath. Three ml of each of these different concentrations were poured into a tube and placed in a relatively dark under the radiation of 808 nm laser with a power density of 1 W/cm2. The temperature change was measured by a digital thermometer equipped with a thermal sensor for 15 min. Figure 9 shows temperature changes over 15 min for nanocomposite solutions at 0.1, 0.5, and 2 mg/ml concentrations, and DI water, with increases of 7.4, 10.9, 13.8, and 5.8 °C, respectively. Data are presented as mean ± SD (n = 3), with error bars indicating standard deviation. Detailed temperature differences for each concentration are provided in Table 1 .Due to the appropriate temperature change and low concentration, the 0.5 mg/ml concentration was determined as the optimal concentration.
Temperature change diagram according to 808 nm laser irradiation time CS-α-Fe2O3 nanocomposite solution in different concentrations. Data are presented as mean ± SD (n = 3). Error bars represent standard deviation.
The photothermal conversion efficiency of the CS-α-Fe2O3 nanocomposite was calculated using Roper’s method under 808 nm laser irradiation. Roper’s equation models the photothermal process as a balance between the heat generated by light absorption and the heat lost to the surroundings, allowing photothermal conversion efficiency to be derived from the steady-state temperature and system parameters48. In the experiment, the temperature of the nanocomposite solution increased from 24 °C to a saturated value of 42.1 °C, while DI water under identical conditions reached only 34.2 °C. Based on this temperature rise and heat transfer analysis, the efficiency was determined to be 7%. Detailed calculations, fitting procedures, and related graphs are provided in the Supplementary Information.
When α-Fe2O3NPs as semiconductors are exposed to laser irradiation, the photocatalytic process is activated, in which the photon energy is used to transfer electrons from the valence band to the conduction band, and at the same time, it creates a similar number of holes in the valence band, which ultimately leads to the formation of an electron-hole pair. Continuing this separation and transfer of charges to the surface leads to oxidation and reduction reactions with the molecules around these α-Fe2O3NPs, which can produce ROS. By studying previous research, the ability of α-Fe2O3NPs to create hydroxyl ROS was determined35. Therefore, methylene blue was used as a hydroxyl radical probe to detect indirectly this type of free radical. For this purpose, in an experiment, 1 mg of NPs powder was dissolved in 1.5 ml of DI water and 0.5 ml of methylene blue (0.2 mg/ml) using an ultrasonic bath. Two similar samples were prepared, and in a relatively dark room, 3 ml of each sample was placed on magnetic stirrers, and only one sample was exposed to an 808 laser with a power density of 1 W/cm2 for 10 min. Then, the methylene blue absorption spectrums of two samples were measured by a spectrometer, and the reduction of the characteristic absorption peak of methylene blue in the sample under laser irradiation compared to the non-irradiated sample at the wavelength of 664 nm was observed. The results are shown in Fig. 10. The electrons and holes created by the photocatalytic process provide the conditions for producing active oxygen species around the α-Fe2O3NPs. In this way, the holes formed in α-Fe2O3NPs lead to the oxidation of the methylene blue molecule, which turns it into an active substance that is ready to react. Also, the electrons on the surface of α-Fe2O3NPs are transferred to oxygen molecules dissolved in water, which produces superoxide radical negative ions. These ions, after reacting with water molecules, can decompose, and consequently, hydroperoxyl radicals and hydroxyl ions are produced. In the end, after reacting hydroperoxyl radicals with water molecules and proton absorption, this process produces hydroxyl radicals as Eq. (3)49:
$$begin{array}{*{20}{c}} {alpha – F{e_2}{O_3}+hnu to alpha – F{e_2}{O_3}+{e^ – }+{h^+}} \ {{h^+}+MB to M{B^{ cdot +}}} \ {{O_2}+{e^ – } to O_{2}^{{ cdot – }}} \ {O_{2}^{{ cdot – }}+{H_2}O to HO_{2}^{ cdot }+O{H^ – }} \ {HO_{2}^{ cdot }+{H_2}O to O{H^ cdot }+{H_2}{O_2}} end{array}$$
(3)
Methylene blue absorption spectrum before and after 808 nm laser irradiation for α-Fe2O3NPs.
To assess the cytotoxicity of the CS-α-Fe2O3 nanocomposite, an MTT assay was carried out on AGS gastric cancer cells under in vitro conditions. This experiment includes three independent biological replicates, each containing five technical replicates. The percentage of relative cell viability was determined by calculating the ratio of treated cell viability to that of untreated control cells. Results are expressed as the mean of three biological replicates (n = 3) ± standard deviation. Statistical analysis was carried out using GraphPad Prism (version 9), with group comparisons performed via analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p value < 0.05 was deemed statistically significant. These Cells were treated with two concentrations of the nanocomposite: 250 ppm (Group 1) and 500 ppm (Group 2). Each concentration was tested under two conditions—with and without irradiation using an 808 nm laser (power density: 1 W/cm², duration: 15 min). Untreated cells were used as the negative control, while a separate control group consisting of cells exposed to laser alone (without the nanocomposite) was included to isolate the impact of irradiation itself.
In Group 1, treatment with 250 ppm of the nanocomposite in the absence of laser exposure resulted in minimal cytotoxicity, with a high cell viability of 97%. However, upon laser irradiation, viability decreased slightly to 88%. In Group 2, treatment with 500 ppm of the nanocomposite without irradiation maintained a similarly high viability of 95%, indicating low inherent toxicity. In contrast, laser-irradiated cells in this group showed a significant decrease in viability to 68%, suggesting a pronounced phototoxic effect. The corresponding results are presented in Fig. 11.
Effect of CS-nanocomposite at two concentrations (250 ppm and 500 ppm), with and without 808 nm laser irradiation, on the viability of AGS cells as determined by the MTT assay. The data are presented as the mean of three independent experiments ± standard deviation. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test (ns = non-significant, ** = p < 0.01, # = statistically significant compared to untreated cells).
The 250 and 500 µg/mL non-laser groups did not show any statistically significant cytotoxicity compared to the control group. Moreover, at the concentration of 250 µg/mL, there was no statistically significant difference between the laser and non-laser treated groups (ns). However, at 500 µg/mL, a statistically significant difference was observed between the laser and non-laser groups. These findings highlight the biocompatibility of the nanocomposite in the absence of laser activation, even at higher concentrations. Conversely, under laser exposure, a concentration-dependent reduction in cell viability was observed, demonstrating the nanocomposite’s potential for application in PTT and PDT therapy.