Fabrication and physicochemical characterization of iron oxide nanoparticles for biomedical applications

Introduction

Nanotechnology has evolved as a transformative discipline in present-day science, offering applications pertaining to innovative solutions across medicine, biotechnology, and materials science (Pathakoti et al., 2018; Malik et al., 2023) [28,21] . Iron oxide nanoparticles (IONPs) are gaining significant interest among other nanomaterials because of their distinct physicochemical and magnetic characteristics, biocompatibility (Xie et al., 2018) [38] , and ease of surface functionalization (Shah et al., 2022) [33] . These qualities enable them to ideal for a variety of biological uses, such as biosensing (Magro et al., 2019; Lee et al., 2020) [20,19] , tissue engineering (Hasan et al., 2018; Friedrich et al., 2021; Truong et al., 2024) [7,5,36] , drug administration (Hussein-Al-Ali et al., 2021) [9] , magnetic resonance imaging (Khmara et al., 2019), and magnetic hyperthermia (Martins et al., 2025) [23] . Superparamagnetic property at nanoscale dimensions allows IONPs, especially magnetite to be modulated under an external magnetic field. This characteristic is particularly useful in diagnostic imaging and targeted drug delivery, as minimum aggregation and precise spatial localization are needed (Jeon et al., 2021) [11] . Additionally, their biodegradability and comparatively low toxicity increase their potential for in-vivo applications. The process of IONP synthesis has remarkable impact on size, surface charge, size distribution, shape and stability of nanoparticles (Janik-Olchawa et al., 2021) [10] . A variety of techniques have been developed including such as co-precipitation, thermal decomposition, hydrothermal synthesis and sol-gel methods (Nikitin et al., 2018; Saqib et al., 2019; Jesus et al., 2024) [27,31,12] . Among all, chemical co-precipitation method is widely accepted considering its convenient nature, affordability, high yield and large scalability. Although, managing particle size distribution and minimizing the nanoparticle aggregation seem to be key challenges for coprecipitation technique (Guardia et al., 2011; Ajinkya et al., 2020) [6,1] . In order to validate the structural, morphological, and functional characteristics of fabricated nanoparticles, characterization is paramount. The dynamic light scattering (DLS), offer insight on particle size, poly dispersity index, zeta potential of nanomaterial and indicates colloidal stability of nanomaterial (Mishra et al., 2014) [25] . Fourier transform infrared spectroscopy (FTIR) is employed to identify functional groups and confirm surface modifications (Stoia et al., 2016) [35] , while scanning electron microscopy (SEM) visualises particle morphology and surface topography.

X-ray diffraction (XRD) analysis ascertained the crystalline structure and phase purity of the nanoparticles (Ali et al., 2016) [2] . Customizing IONPs for enhanced therapeutic efficacy and biological compatibility has garnered more emphasis in recent years. Their stability, circulation time, and targeting ability can be dramatically improved by surface modification using polymers, surfactants, or bioactive compounds (Natarajan et al., 2019) [26] . Therefore, the present study primarily targeted synthesis of iron oxide nanoparticles using an appropriate methodology and their thorough characterisation incorporating DLS, FTIR, SEM, and XRD techniques. The results are intended for the development of efficient and biocompatible nanomaterials for biological and clinical applications.

Materials & Methods Materials

All chemical reagents utilized in the current study were of analytical grade, which ensures high purity. Anhydrous ferric chloride (FeCl 3 ) and ferrous sulphate heptahydrate (FeSO 4 .7H 2 O) were procured from Molychem and HiMedia Laboratories Pvt. Ltd., respectively. Additionally, ammonium hydroxide solution (18-20%) was employed as precipitating reagent and absolute ethanol was used as solvent.

Fabrication of iron oxide nanoparticles

The synthesis of iron oxide nanoparticles was conducted using the chemical coprecipitation method, following the methods outlined by Dehghanpour (2020) [4] together with a couple of changes. Anhydrous ferric chloride and ferrous sulphate heptahydrate were utilized as iron precursors. For this 1.62 gm of ferric chloride was added in 5 mL distilled water to prepare 2M solution and 1.39 gm of ferrous sulphate was added into another 5 mL of distilled water to obtain 1M solution. Then each solution were transferred into a conical flask and mixed with the help of magnetic stirrer at the speed of 1000 rpm, 80 °C for one hour. After that, 2M solution of ammonium hydroxide was incorporated drop by drop until the solution has turned completely black in colour. The resultant mixture was stirred vigorously for next 60 minutes. The mixture was subsequently allowed to settle at ambient room temperature, following that the supernatant was removed. The black sediment was subsequently retrieved and washed thrice with absolute ethanol and thrice with double distilled water, then dried in a hot air oven at 60 °C for 4-6 hours.

Characterization of iron oxide nanoparticle Estimation of particle size, zeta potential and poly dispersity index (PDI)

Determination of particle size, PDI and zeta potential was carried out on the basis of dynamic light scattering technique. In our study SZ-100, Zetasizer (HORIBA) instrument was used (Perissé Moreira and Grasseschi, 2022) [29] .

Surface morphology of iron oxide nanoparticles

Scanning electron microscopy (SEM) is a sophisticated imaging technique extensively employed for the morphological characterization of nanoscale materials. The analysis was carried out using a Hitachi UHR FE-SEM SU8010 instrument. The dried powder samples were evenly dispersed onto a carbon-coated aluminium stub and then coated with a thin layer of gold using a sputter coater. The specimens were subsequently examined under high-vacuum conditions at an accelerating voltage of 10 kV and micrographs were obtained at different magnifications (Selvam, et al., 2024) [32] .

Fourier-transform infrared spectroscopy (FT-IR) of iron oxide nanoparticles

Fourier-transform infrared spectroscopy (FT-IR) is a widely applied analytical method for recognizing and characterizing functional groups and chemical bonds present in given sample. The FT-IR spectra of iron oxide nanoparticle were obtained within the spectral range of 4000-400 cm⁻¹ employing a PerkinElmer Spectrum equipment. A pellet of iron oxide nanoparticle powder sample was prepared with potassium bromide (KBr) prior to analysis (Zakariya, et al., 2022) [40] .

X-Ray Diffraction (XRD) analysis of iron oxide nanoparticles

X-ray diffraction (XRD) examination was conducted to assess the crystalline structure, phase composition, and crystallite size of the produced iron nanoformulation and iron-zinc nanocomposite. The diffraction patterns have been obtained through a PANalytical X-ray diffractometer employing Cu K-α₁ radiation (λ = 1.5406 Å), functioning at 45 kV and 40 mA. The finely prepared powdered specimens were meticulously affixed to the sample holder to guarantee a smooth and uniform surface before examination. The acquisition of data was conducted within a 2θ scanning range of 10° to 80°. The obtained diffraction peaks were analyzed against standard reference patterns from the Joint Committee on Powder Diffraction Standards (JCPDS) database to identify the crystalline phases present (Saqib, et al., 2019) [31] .

Results

Synthesis of iron oxide nanoparticles

The synthesized iron oxide nanoparticle appeared as black colour fine powder. From one batch we obtained approximately 800 mg iron oxide nanoparticles. These prepared iron oxide nanoparticle showed characteristic magnetism property as depicted in Figure 1

Characterization of iron oxide nanoparticle Particle size, zeta potential and poly dispersity index (PDI)

The synthesized iron oxide nanoparticle showed average particle size around 99 nm. The poly dispersity index was around 0.416 and zeta potential found-45.9 mV (Figure 2 and Figure 3).

Surface morphology

Scanning electron microscopy (SEM) images of iron oxide nanoparticle exhibited almost spherical shape (Figure 4). However, few particles showed irregular shaped and aggregations. Surface of iron oxide particles displayed nearly smooth texture. The size and shape of prepared nanoparticles mostly uniform.

Fourier-transform infrared spectroscopy (FT-IR)

The FT-IR spectrum of iron oxide nanoparticle is depicted in Figure 5. The FT-IR spectrum of iron oxide nanoparticles revealed presence of distinct prominent peaks at 3411.83 cm⁻¹, 1628.27 cm⁻¹ and 586.92 cm⁻¹ which are associated with the presence of hydroxyl group (O-H), water molecules (H-O-H) and metal oxide stretching (Fe-O). Few minor peaks were also recorded around 1402 cm⁻¹ and 1109 cm⁻¹ showed presence of organic solvent residue and peak and 797 cm⁻¹ was indicated the presence of metal oxide related stretching.

X-Ray Diffraction (XRD)

The iron oxide nanoparticles displayed six distinct diffraction peaks on XRD analysis. These different diffraction peaks were located at 2θ ≈ 30.17°, 35.42°, 43.56°, 53.12°, 57.05°, and 62.99°, corresponding to the (220), (311), (400), ( 422), (511), and (440) planes of spinel iron oxide structure (Figure 6). [22,16,14] . However, some studies (Khalil et al., 2015; Mazrouaa et al., 2019) [13,24] reported the use of sodium hydroxide as alkaline reagent instead of ammonium hydroxide. Zeta potential measurement and dynamic light scattering (DLS) were used to determine the surface charge and particle size of the synthesized IONPs. The successful synthesis of nanoscale particles having a relatively narrow size range was recorded hydrodynamic diameter of 99 nm. Sheel et al. (2020) [34] , also reported the similar particle size. A wide range of the particle size ranging from 5 nm to 150 nm was also reported in various studies (Kovrigina et al., 2022) [18] , which might be attributed to difference in protocol employed for synthesis of IONP. The nanoscale dimension is crucial for potential application in biomedical, as it facilitates the higher bioavailability, longer circulation time and enhanced cellular uptake (Salehirozveh et al., 2024) [30] .

The formed IONP showed around-45 mV zeta potential, suggesting a very stable colloidal dispersion. Zeta potential is key indicator of stability of nanoparticle and higher than±25 mV often reflects strong electrostatic repulsion between particles and prevents aggregation. These findings are supported by previous study (Constantino et al., 2024) [3] , who also found high negative zeta potential (-63 mV). [37, [8] , who also reported similar characteristic peak of Fe-O bond.

Although, this peak generally recorded between 450-600 cm⁻¹ regions.

The XRD pattern of synthesized IONP showed six characteristic diffraction peaks, which ensure the presence crystalline iron oxide core and high phase purity. Similarily, six distinct diffraction peaks were also recorded (Yoonus et al., 2021; Kiwumulo et al., 2022) [39,17] at 2θ ≈ 30°, 35°, 43°, 53°, 57° and 63°.

Conclusion

Iron oxide nanoparticles (IONP) were effectively fabricated using simple and economic chemical co-precipitation method. Physicochemical characterisation confirmed their nanoscale size, uniform size distribution, good stability and crystalline nature along with distinctive Fe-O bond. These attributes suggest that the produced IONPs are suitable for biomedical uses including drug delivery and imaging. However, further studies on biocompatibility and in vivo efficacy are required to validate their clinical use.

References

1. Ajinkya N, Yu X, Kaithal P, Luo H, Somani P, Ramakrishna S. Magnetic iron oxide nanoparticle (IONP) synthesis to applications: present and future. Materials. 2020;13(20):4644.
2. Ali A, Zafar H, Zia M, ul Haq I, Phull AR, Ali JS, et al. Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnol Sci Appl. 2016;9:49-67.
3. Constantino JS, Mesquita IDD, Moraes Segundo JD, Moreira Filho RN, Araújo ABD, Ferreira MV, et al. Antiviral and nontoxic dermal iron oxide nanoparticle/biopolymer coatings for cotton fabric. ACS Appl Nano Mater. 2024;7(12):13991-14004.
4. Dehghanpour HR. pH, molar ratio of ferrous to ferric ions and surfactant presence effects on physical properties of iron oxide nanoparticles generated by coprecipitation method. J Coord Chem. 2020;73(24):3452-3464.
5. Friedrich RP, Cicha I, Alexiou C. Iron oxide nanoparticles in regenerative medicine and tissue engineering. Nanomaterials. 2021;11(9):2337.
6. Guardia P, Labarta A, Batlle X. Tuning the size, the shape, and the magnetic properties of iron oxide nanoparticles. J Phys Chem C. 2011;115(2):390-396.
7. Hasan A, Morshed M, Memic A, Hassan S, Webster TJ, Marei HES. Nanoparticles in tissue engineering: applications, challenges and prospects. Int J Nanomedicine. 2018;13:5637-5655.
8. Hussain A, Yasar M, Ahmad G, Ijaz M, Aziz A, Nawaz MG, et al. Synthesis, characterization, and applications of iron oxide nanoparticles. Int J Health Sci. 2023;17(4):3.
9. Hussein-Al-Ali SH, Hussein MZ, Bullo S, Arulselvan P. Chlorambucil-iron oxide nanoparticles as a drug delivery system for leukemia cancer cells. Int J Nanomedicine. 2021;16:6205-6216.
10. Janik-Olchawa N, Drozdz A, Ryszawy D, Pudelek M, Planeta K, Setkowicz Z, et al. The influence of IONPs core size on their biocompatibility and activity in in vitro cellular models. Sci Rep. 2021;11(1):21808.
11. Jeon M, Halbert MV, Stephen ZR, Zhang M. Iron oxide nanoparticles as T1 contrast agents for magnetic resonance imaging: fundamentals, challenges, applications, and prospectives. Adv Mater. 2021;33(23):1906539.
12. Jesus J, Regadas J, Costa B, Carvalho J, Pádua A, Henriques C, et al. Green sol-gel synthesis of iron oxide nanoparticles for magnetic hyperthermia applications. Pharmaceutics. 2024;16(12):1578.
13. Khalil MI. Co-precipitation in aqueous solution synthesis of magnetite nanoparticles using iron (III) salts as precursors. Arab J Chem. 2015;8(2):279-284.
14. Khanam J, Ahmed MF, Zaman SKM, Sharmin N, Ahmed S. Synthesis of nano-sized magnetic iron oxide by a simple and facile co-precipitation method. Bangladesh J Sci Ind Res. 2022;57(2):67-76.
15. Khmara I, Strbak O, Zavisova V, Koneracka M, Kubovcikova M, Antal I, et al. Chitosan-stabilized iron oxide nanoparticles for magnetic resonance imaging. J Magn Magn Mater. 2019;474:319-325.
16. Kim JH, Kim SM, Kim YI. Properties of magnetic nanoparticles prepared by co-precipitation. J Nanosci Nanotechnol. 2014;14(11):8739-8744.
17. Kiwumulo HF, Muwonge H, Ibingira C, Lubwama M, Kirabira JB, Ssekitoleko RT. Green synthesis and characterization of iron-oxide nanoparticles using Moringa oleifera. BMC Res Notes. 2022;15(1):149.
18. Kovrigina E, Chubarov A, Dmitrienko E. High drug capacity doxorubicin-loaded iron oxide nanocomposites for cancer therapy. Magnetochemistry. 2022;8(5):54.
19. Lee DY, Kang S, Lee Y, Kim JY, Yoo D, Jung W, et al. PEGylated bilirubin-coated iron oxide nanoparticles as a biosensor for magnetic relaxation switching-based ROS detection in whole blood. Theranostics. 2020;10(5):1997.
20. Magro M, Baratella D, Miotto G, Frömmel J, Šebela M, Kopečná M, et al. Enzyme self-assembly on naked iron oxide nanoparticles for aminoaldehyde biosensing. Amino Acids. 2019;51(4):679-690.
21. Malik S, Muhammad K, Waheed Y. Emerging applications of nanotechnology in healthcare and medicine. Molecules. 2023;28(18):6624.
22. Mandel K, Hutter F, Gellermann C, Sextl G. Synthesis and stabilisation of superparamagnetic iron oxide nanoparticle dispersions. Colloids Surf A. 2011;390(13):173-178.
23. Martins C, Rolo C, Cacho VR, Pereira LC, Borges JP, Silva JC, et al. Enhancing the magnetic properties of superparamagnetic iron oxide nanoparticles using hydrothermal treatment for magnetic hyperthermia application. Mater Adv. 2025;6(5):1726-1733.
24. Mazrouaa AM, Mohamed MG, Fekry M. Physical and magnetic properties of iron oxide nanoparticles with a different molar ratio of ferrous and ferric. Egypt J Pet. 2019;28(2):165-171.
25. Mishra D, Arora R, Lahiri S, Amritphale SS, Chandra N. Synthesis and characterization of iron oxide nanoparticles by solvothermal method. Prot Met Phys Chem Surf. 2014;50(5):628-631.
26. Natarajan S, Harini K, Gajula GP, Sarmento B, NevesPetersen MT, Thiagarajan V. Multifunctional magnetic iron oxide nanoparticles. BMC Mater. 2019;1(1):2.
27. Nikitin AA, Shchetinin IV, Tabachkova NY, Soldatov MA, Soldatov AV, Sviridenkova NV, et al. Synthesis of iron oxide nanoclusters by thermal decomposition. Langmuir. 2018;34(15):4640-4650.
28. Pathakoti K, Manubolu M, Hwang HM. Nanotechnology applications for environmental industry. In: Handbook of nanomaterials for industrial applications. Elsevier; 2018. p. 894-907.
29. Perissé Moreira M, Grasseschi D. Automation of a lowcost device for flow synthesis of iron oxide nanoparticles. J Nanopart Res. 2022;24(5):93.
30. Salehirozveh M, Dehghani P, Mijakovic I. Synthesis, functionalization, and biomedical applications of iron oxide nanoparticles (IONPs). J Funct Biomater. 2024;15(11):340.
31. Saqib S, Munis MFH, Zaman W, Ullah F, Shah SN, Ayaz A, et al. Synthesis, characterization and use of iron oxide nanoparticles for antibacterial activity. Microsc Res Tech. 2019;82(4):415-420.
32. Selvam K, Sudhakar C, Senthilkumar B, Sakthivel V, Ragu Prasath A, Sangameshwaran V. Green synthesis and characterisation of iron oxide nanoparticle using Ziziphus oenoplia fruit extract. Waste Biomass Valor. 2024;15(11):6415-6429.
33. Shah ST, Chowdhury ZZ, Johan MRB, Badruddin IA, Khaleed HMT, Kamangar S, et al. Surface functionalization of magnetite nanoparticles. Molecules. 2022;27(3):789.
34. Sheel R, Kumari P, Panda PK, Ansari MD, Patel P, Singh S, et al. Oxidative stress and apoptosis modulated in vivo biocompatibility of antibacterial iron oxide nanoparticles with zebrafish. Environ Pollut. 2020;267:115482.
35. Stoia M, Istratie R, Păcurariu C. Investigation of magnetite nanoparticles stability in air by thermal analysis and FTIR spectroscopy. J Therm Anal Calorim. 2016;125(3):1185-1198.
36. Truong TT, Mondal S, Doan VHM, Tak S, Choi J, Oh H, et al. Precision-engineered metal and metal-oxide nanoparticles for biomedical imaging and healthcare applications. Adv Colloid Interface Sci. 2024;332:103263.
37. Winiarczyk K, Gac W, Góral-Kowalczyk M, Surowiec Z. Magnetic properties of iron oxide nanoparticles with a DMSA-modified surface. Hyperfine Interact. 2021;242(1):48.
38. Xie W, Guo Z, Gao F, Gao Q, Wang D, Liaw BS, Cai Q, Sun X, Wang X, Zhao L. Shape-, size-, and structure-controlled synthesis and biocompatibility of iron oxide nanoparticles for magnetic theranostics. Theranostics. 2018;8(12):3284.
39. Yoonus J, Resmi R, Beena BJMTP. Evaluation of antibacterial and anticancer activity of green synthesized iron oxide (α-Fe₂O₃) nanoparticles. Mater Today Proc. 2021;46:2969-2974.
40. Zakariya NA, Majeed S, Jusof WHW. Investigation of antioxidant and antibacterial activity of iron oxide nanoparticles synthesized from Penicillium spp. Sensors Int. 2022;3:100164.