Simple rapid stabilization method through citric acid modification for magnetite nanoparticles

A highly stable and magnetized citric acid (CA)-functionalized iron oxide aqueous colloidal solution (Fe3O4@CA) was synthesized by using a simple and rapid method of one-step co-participation via a chemical reaction between Fe3+ and Fe2+ in a NaOH solution at 65 °C, followed by CA addition to functionalize the Fe3O4 surface in 25 min. The NPs were synthesized at lower temperatures and shortened time compared with conventional methods. Surface functionalization is highly suggested because bare Fe3O4 nanoparticles (Fe3O4 NPs) are frequently deficient due to their low stability and hydrophilicity. Hence, 19 nm-sized Fe3O4 NPs coated with CA (Fe3O4@CA) were synthesized, and their microstructure, morphology, and magnetic properties were characterized using X-ray diffraction, transmission electron microscopy, Zeta potential, Fourier transform infrared spectroscopy, and vibrating sample magnetometer. CA successfully modified the Fe3O4 surface to obtain a stabilized (homogeneous and well dispersed) aqueous colloidal solution. The Zeta potential value of the as-prepared Fe3O4@CA increases from − 31 to − 45 mV. These CA-functionalized NPs with high magnetic saturation (54.8 emu/g) show promising biomedical applications.

Fe 3 O 4 NPs with a grain size of smaller than 20 nm display superparamagnetic behavior at high temperatures but exhibit no coercivity and remanence at room temperature [1][2][3][4] . These particles are extensively utilized for several biomedical and in vivo applications [5][6][7][8][9] . Fe 3 O 4 NPs, a well-known ferrofluid, has been expansively analyzed, particularly their colloidal dispersion and many potential biomedical applications. The surface of magnetite particles is modified by different coating agents, including protein 10 , methoxypoly (ethylene glycol) 11 , dextran 12 , chitosan 13 , and silica coating 14 , to enhance their performance. Controlling the sizes and dispersion of NPs in preferred solvents is technologically challenging due to difficulties faced in their fabrication and handling for biomedical applications, including their clustering/aggregation, homogeneity, hydrophilicity, and biocompatibility 15,16 . The high surface energies of NPs are attributed to their large surface to volume ratio. NPs tend to aggregate to minimize total surface energy, which exceeds 0.1 N/m for metal oxide surfaces 17 .
Proper functionalization of NP surface and solvent selection are critical to attain adequate repelling interactions between the NPs to inhibit agglomeration/accretion and improve the thermodynamic stability of the colloidal solution. The surface of Fe 3 O 4 dispersed in aqueous media via citric acid adsorption can be functionalized by utilizing the coordination of one or two carboxylate functionalities of the citric acid depending on the steric necessity and curvature of the surface 18 . Carboxylates significantly affect the development of Fe 3 O 4 NPs and their magnetic characteristics. Surface modification of aqueous magnetic NPs by using heavy chain fatty acid or thiol is one of the methods to increase the stability of NP suspension 19 . Co-precipitation is typically used to synthesize water-stable Fe 3 O 4 NPs and considered as the simplest, most cost-effective technique requiring the lowest temperature 20 . However, its main drawbacks are the agglomeration, broad size distribution, poor Zeta potential values of NPs. Fe 3 O 4 NPs also lack good colloidal stability and have inadequate repulsive forces to prevent agglomeration. The poor colloidal stability and broad size distribution can be attributed to the reaction time and temperature during co-precipitation. To overcome these problems, the Fe 3 O 4 NPs must be stabilized and their size distribution must be reduced by modifying their surfaces with biocompatible materials, in addition to controlling the synthesis procedures. Nevertheless, most of aqueous stabilized Fe 3  To the extent of our knowledge, the stability of Fe 3 O 4 @CA NPs has not been reported. Therefore, this study aims (1) to synthesize a highly stable and magnetized Fe 3 O 4 @CA aqueous colloidal solution by employing a onestep, fast, and straightforward route (with shortened time and lower temperature than conventional methods) and systematically controlling and manipulating the flow of the reaction procedure and (2) to develop surface functional groups on magnetic NP derivatization through a one-step process.

Materials and methods
Materials. Ferric chloride (FeCl 3 ·6H 2 O, 99%), ferrous chloride (FeCl 2 ·4H 2 O, 99%), and sodium hydroxide (NaOH) were acquired from Sigma-Aldrich, and citric acid (CA) were purchased from Merck. preparation fe 3 o 4 @CA. Fe 3 O 4 NPs were synthesized through the co-precipitation of ferrous (Fe 2+ ) and ferric (Fe 3+ ) with sodium hydroxide (NaOH). FeCl 2 .4H 2 O (2.5 g) and FeCl 3 .6H 2 O (4.0 g) were dissolved in 180 mL of distilled water under nitrogen gas. Following the complete dissolution of the mixture at room temperature, 50 mL of sodium hydroxide was drop-wise added to the reaction mixture, which was mechanically stirred at 650 rpm and kept for 10 min at 65 °C under continuous vigorous stirring. For the prevention of Fe 3 O 4 NP agglomeration, 150 mL of CA was added to the reaction mixture, which was then stirred for 10 min (65 °C). Fe 3 O 4 @CA NPs were collected through a permanent magnet and thoroughly rinsed four times with distilled water to eliminate unreactive or inert impurities. Finally, the Fe 3 O 4 @CA NPs were redispersed in the distilled water after sonication for 5 min, and the resulting suspension (Fe 3 O 4 @CA) responded to an external magnetic field as shown in Fig. 1. characterization of fe 3 o 4 @CA. X-ray diffraction (XRD) patterns were obtained using an X-ray diffractometer (PANalytical X'pert PRO MRD PW 3040) with CuKa (λ = 1.54050 Å). The size of Fe 3 O 4 NPs and Fe 3 O 4 @Au CSNPs were obtained by transmission electron microscopic (TEM) using a Zeiss Libra 120 at 100 kV. Particle size distribution was measured using ImageJ software. The stability (Zeta potential) of Fe 3 O 4 @CA NPs was described using a dynamic light scattering (DLS) instrument (ZETASIZER Nanoseries Model ZEN 3600, Malvern Instruments). The surface functional groups of Fe 3 O 4 @CA NPs were determined by Fourier transform infrared spectroscopy (PERKIN ELMER System 2000 FT-IR). Magnetic properties were evaluated using vibrating sample magnetometer (VSM, DMS MODEL 8810).

Results and discussion
Fe 3 O 4 NP surfaces were functionalized via CA adsorption, which occurs by coordinating one or two of the carboxylate functionalities depending on the need for steric repelling to stabilize the ferrofluids and the curvature or morphology of the surface. Nonetheless, a minimum of one carboxylic acid group is exposed to the solvent, thus accounting for the surface charging. The presence of a carboxylic group surface ligand offers the possibility of developing bonds with proteins, fluorescent dyes, and hormone linkers to facilitate precise targeting in biological systems. The one-step modification of the superparamagnetic Fe 3 O 4 NP surface is presented in Fig. 2, and the as-prepared Fe 3 O 4 NPs were subsequently stabilized with CA to prevent agglomeration.
The XRD spectra (Fig. 3) (Fig. 4). Figure 4a shows the TEM image of Fe 3 O 4 prior to CA modification. A slightly important change in Fe 3 O 4 agglomeration was induced by CA. From the histogram in Fig. 4c, the average size of the monodispersed Fe 3 O 4 @CA NPs is approximately 19 nm. The Fe 3 O 4 @CA NPs are spherical in shape with a narrow size distribution after CA modification, particularly at stable synthesis conditions. The TEM images of the CA-functionalized superparamagnetic Fe 3 O 4 NPs show semi-spherical shaped particles and monodispersion. Previous studies used co-precipitation to synthesize CA-coated Fe 3 O 4 and produced agglomerated NPs with average sizes 51 28 , 50 32 , 25, 33 and 22 nm 34 , which might be due to the high reaction temperature 35 . Figure 5 presents the stability of Fe 3 O 4 after CA modification to show the role of CA on the stability of Fe 3 O 4 NPs. The Zeta potential magnitude of Fe 3 O 4 NPs was measured immediately after the synthesis of the particles, followed by CA injection on the colloidal Fe 3 O 4 NPs. Dispersion stability can be defined in relation to the Zeta potential value (mV): 0 to ± 5 can cause the rapid agglomeration and precipitation of NP suspension, ± 10 to ± 30 is responsible for the threshold of delicate dispersion, ± 30 to ± 40 denotes the moderate stability of colloidal NPs [36][37][38][39] , and ± 40 to ± 60 indicates the excellent stability of NP suspension referred to as a high charge on their surface 40 . The measured results indicate that Zeta potential improves from − 31.3 to − 45.3 mV (Fig. 5a, b), which is higher than the reported values [41][42][43] and is attributed to the three carboxylate groups of citrate that dissociate  www.nature.com/scientificreports/ and strongly bind with Fe 3 O 4 NP surface 18 . In addition, the negative charge of the Zeta potential is due to the electrostatic stabilization provided by the strong adsorption of citrate ions on NP surface. The Zeta potential of CA-coated Fe 3 O 4 has not been reported. The increase in the measured Zeta potential revealed that CA is absorbed onto the Fe 3 O 4 NP surface, thus resulting in a highly negative surface charge. The presence of carboxylate group is confirmed by monitoring the Zeta potential for Fe 3 O 4 NPs. However, this study characterized and analyzed only the results for a moderately polydispersed sample. The Fourier transform-infrared spectroscopy (FT-IR) spectrum of bare Fe 3 O 4 has been reported 28,29 . This study aimed to prove the presence of CA on the Fe 3 O 4 surface. FT-IR spectra of CA and Fe 3 O 4 @CA NPs are illustrated in Fig. 6a and b. The spectrum peak was assigned to the CA-coated Fe 3 O 4 NPs. The broad band spectrum at 3,384 cm −1 can be referred to as the OH band groups and to the traces of molecular water. The 1722 cm −1 spectrum peak of CA is due to the symmetric C=O stretching from the COOH group. This peak display shifts to a lower wavelength at approximately 1615 cm −1 for the carboxylic group (R-OOH) of the Fe 3 O 4 @CA. The peak  The magnetic properties of Fe 3 O 4 @CA were determined by VSM analysis at room temperature. The magnetization saturation (emu/g) as a function of the applied magnetic field (Oe) is illustrated in Fig. 7. The magnetization curve shows that the Fe 3 O 4 @CA NPs exhibit a superparamagnetic behavior and magnetic saturation (Ms) of approximately 54.8 emu/g, which is higher than that in previous studies 25,33 (Table 1) possibly due to the low Fe oxidation state. No hysteresis was observed, and the behavior was completely reversible at 300 K. Neither coercivity nor remanence was observed. Arefi et al. 28 . reported that the Ms of bare Fe 3 O 4 is reduced after being coated with CA. Alonso et al. 23 synthesized Fe 3 O 4 NPs with high crystallinity of approximately 35 nm and high Ms of 65 emu/g by using thermal decomposition. The high Ms value is attributed to the large particle size of Fe 3 O 4 45 . Therefore, the Ms of Fe 3 O 4 NPs decreases with their reduced particle size due to the increase in surface spin disorder 46,47 . In this case, the size reduction to the nanoscale (below 20 nm) for spherical single-component Fe 3 O 4 greatly influences the magnetic ordering of surface spins, namely, a high degree of disordered surface spins of Fe 3 O 4 NPs. Spherical Fe 3 O 4 NPs could develop surface spin disorder through energy minimization. The disordered surface spins are highly anisotropic, which is in line with the increase in their effective magnetic anisotropy.
For biomedical applications such as in hyperthermia and magnetic resonance imaging (MRI), NPs must have a uniform particle size, exhibit superparamagnetism, and possess high Ms. The as-synthesized Fe 3 O 4 @CA has a high magnetic response, which is preferable for biomedical applications 28 . Our method shows an advantage of having a simple and rapid route to synthesize highly stable (− 45.3 mV), monodispersed, and superparamagnetic Fe 3 O 4 @CA (19 nm) compared with conventional techniques. We developed a simple and rapid synthesis route for highly stable and superparamagnetic Fe 3 O 4 @CA through co-precipitation. The proposed method requires simple equipment and cheap materials such as a magnetic stirrer, and the processing time was 25 min at 65 °C. The NPs were achieved at lower temperature, simpler process, and shorter time compared with conventional methods. XRD, TEM, Zeta potential, FT-IR, and VSM were employed to characterize the microstructure and morphology of the synthesized NPs. The presence of carboxylate group is confirmed by FTIR analysis, and the Zeta potential for Fe 3 O 4 particles was monitored. The Zeta potential value of as-prepared Fe 3 O 4 @CA increased from − 31.3 to − 45.3 mV. Finally, these NPs are important for several biomedical applications due to their small size, stability, and superparamagnetic behavior.