Stable aqueous dispersion of superparamagnetic iron oxide nanoparticles protected by charged chitosan derivatives

The synthesis of SPIONs was carried out in an aqueous solution. First, iron salts in molar ratio Fe(III):Fe(II) = 2:1, (0.1622 g FeCl₃·6H₂O and 0.0596 g FeCl₂·4H₂O), were dissolved in 50 mL of aqueous solution of CCh (c = 1 or 3 g L⁻¹) obtaining the pH value of ca. 1.5. The solution was deoxygenated by purging with argon and sonicated (Sonic-6, Polsonic, 480 W, 1 s pulse per every 5 s) for 10 min in a thermostated bath at 20 °C. Afterward, 5 mL of 5 M NH_3(aq) was added dropwise, and the solution was further sonicated for 30 min. The obtained suspension was filtered by cellulose syringe filters (0.2 μm). Finally, the SPIONs were purified by magnetic chromatography. Toward this purpose, suspension of the SPIONs was injected into the column filled with ferromagnetic wires and exposed to high magnetic field. Such loaded column was flushed with water. Then, the magnets were removed, and the purified SPIONs were eluted from the column. SPION1 were obtained at the CCh concentration equal to 1 g L⁻¹ and SPION3 at the concentration 3 g L⁻¹, respectively.

To obtain the negatively charged SPIONs, the synthesized nanoparticles were additionally coated with anionic derivative of chitosan (ACh) using LbL method. Aqueous solution of ACh (10 mL, c = 1 g L⁻¹ in 0.1 M NaCl) was added to the SPION1 suspension (4 mL) and sonicated continuously for 10 min at 25 °C. The coated nanoparticles, SPION1/ACh, were purified using magnetic chromatography, as described above.

The negatively charged SPIONs with biocompatible thin polymer coating were synthesized in a two-step procedure. In the first step, the iron oxide nanoparticles were formed from a mixture of FeCl₃ and FeCl₂ salts by reacting with ammonia in the presence of cationic derivative of chitosan (CCh). Chitosan was intentionally modified with charged quaternary amine groups to achieve its solubility in the basic solution of the reaction mixture upon addition of ammonia. The role of this polymer was to complex the iron ions and thus to limit the growth of the nanoparticles during co-precipitation. When polyanions, such as poly(acrylic acid), are used for this purpose, they rather form gels with iron(III) ions (Yokoi et al. 1993). The parameters of the SPIONs synthesis were carefully optimized varying the conditions, such as concentration of the reagents, temperature, stirring method and speed, and sonication protocol, to obtain the nanoparticles of preferred properties. Sonication was applied in all experiments because it was found to be more effective in the avoidance of nanoparticle aggregation than mechanical or magnetic stirring. Owing to the positive correlation observed between the reaction temperature and the size, as well as the size distribution of SPIONs, the reaction medium was cooled and thermostated at 20 °C. Also, the appropriate sonication protocol was applied to prevent the rapid heating of the sample usually occurring during continuous sonication. Magnetic chromatography applied here enabled efficient and easy purification of SPIONs from the remaining reagents. The presence of the cationic polymer, CCh, during the co-precipitation leads to fabrication of stable aqueous dispersion of small aggregates of SPIONs required for biomedical applications. This can be explained considering the fact that SPIONs formed in a presence of CCh exhibited highly positive zeta potential—greater than 40 mV for both polymer concentrations used during the synthesis (see Table 1). There is practically no precipitation in the suspension of SPIONs after a few weeks' storage time. For longer times, some aggregation can be noticed; however, good dispersion may be easily recovered by applying the short sonications. While the other parameters of SPIONs also did not significantly differ for both concentrations of CCh used in the synthesis, only SPION1 were chosen for further modifications.

Such positively charged nanoparticles obtained, in the second step of the fabrication process, were coated with ACh leading to SPION1/ACh. The LbL technique based on the alternating adsorption of oppositely charged polyelectrolytes was employed here. Thanks to high positive surface charge of SPION1 covered by CCh and the same backbone of the oppositely charged ACh, the adsorption was very efficient as indicated by inversion of the zeta potential to the value reaching even less than −40 mV for SPION1/ACh (see Table 1). This value also ensures the stability of the aqueous dispersion of those coated SPIONs.

The efficiencies of the synthetic procedures were determined based on the iron content in the final, purified suspensions. The theoretical content of iron was calculated based on the feed composition of the reaction mixture. The efficiencies of 24 % for SPION1 and 39.9 % for SPION3 reflected rather, not the level of conversion, but the loss of material in the form of larger aggregates during the purification procedure (filtering and magnetic chromatography).

TEM was applied to measure the shape and size of the synthesized nanoparticles (see Fig. 1). The image shows well-separated nanoparticles within a small aggregate. The core sizes of SPION1 were found to be very small with an average diameter being equal to 11.9 ± 1.7 nm (see Fig. 2). The size of the nanoparticles upon coating with ACh remained practically the same (d = 10.1 ± 2.9; Fig. 2). This can be explained considering that ACh layer is ultrathin, which is typical for LbL films formed as a result of electrostatic self-assembly of polyelectrolytes used (Crouzier et al. 2010), and it does not provide sufficiently efficient contrast in TEM. Relatively low standard deviation values indicate narrow distributions of the nanoparticle diameters as preferred for biological applications.

DLS measurements have shown that in aqueous dispersion, nanoparticles exist in the form of small aggregates. The data indicated that hydrodynamic diameters of the obtained SPION1 and SPION1/ACh aggregates are generally less than 100 nm (see Fig. 3). The average diameter for negatively charged SPION1/ACh seems to be slightly smaller than the size of the precursor SPION1. This can be ascribed to some differences in the thicknesses of the electric double layers for both the studied samples (e.g., slightly different zeta potential), which contribute to the total hydrodynamic size measured by DLS. Moreover, the measured diameters may be also affected by surface-bound polymeric chains which slow the diffusion and increase the apparent size of the particles/aggregates. Nevertheless, DLS results are not able provide a direct support for successful coating of the SPION1 since the determination of the diameters of these particles can be significantly affected by other parameters, including also the dynamic character of the formed aggregates. The obtained values are reasonable taking into account the hydration of the chitosan coated and highly charged nanoparticles. The coating is evidenced by measurements of zeta potential. Moreover, it has been reported that relaxivity values (see later for details) of such clusters are greatly improved when compared to the values for isolated nanoparticles. That is essential for MRI applications (Berret et al. 2006; Matsumoto and Jasanoff 2008).

HREM images for SPION1 (see Fig. 4) were also used for determination of the crystal structure of the obtained nanoparticles. It appears that the nanoparticles are mostly composed of single domains, which also implies their preferred magnetic properties (see later). The interplanar crystal spacings were measured and compared with crystal spacings for the iron (II, III) oxides (see Table 2). It has been reported that under the anaerobic conditions applied in the co-precipitation method, Fe₃O₄ is preferentially formed (Gupta and Gupta 2005). However, the crystallographic structures of Fe₃O₄ (magnetite) and Fe₂O₃ (maghemite) are very similar, and it is very difficult to confirm the presence of exclusively one phase in the synthesized SPIONs. Nevertheless, one can conclude that the presence of CCh during the precipitation does not negatively affect the structures of SPIONs and their magnetic properties (see later) leading to the formation of highly crystalline iron oxide nanoparticles.

To confirm the presence of chitosan layers and to determine their content in the nanoparticles, thermogravimetric analysis (TGA) was performed. Figure 5 shows TGA curves for SPION1 and for CCh as the only component of the nanoparticles that decompose in the applied temperature range. The observed behavior is similar to the one reported for ferrogels containing chitosan (Hernández et al. 2009). Based on the TGA data, it was found that the content of CCh in SPION1 sample was around 32 %. That seems to be a reasonable value that is necessary to densely coat the nanoparticles, while it is not too high to significantly decrease the magnetization of this hybrid material (see later).

The interactions between CCh polymer and iron in the hybrid SPION1 were later studied using FTIR (see Fig. 6). There are characteristic peaks at 1,479 cm⁻¹ for the methyl band of GTMAC, at 1,572 cm⁻¹ for the primary amine group (Cho et al. 2006) and at 1,639 cm⁻¹ for the amide I band (Wang et al. 2008; Hernández et al. 2009) in the spectrum of CCh. The similarity of the overall shape of the FTIR spectra for SPION1 and CCh confirm again the presence of CCh in the hybrid nanoparticles. Moreover, the shift of the peak at 1,564 cm⁻¹ indicates the interaction of the primary amine group with the iron ions and the creation of amino complexes (Hernández et al. 2009) as postulated above.

The magnetic properties of the fabricated SPIONs were evaluated using various methods. The magnetization in a function of magnetic field was measured for SPION1. The shape of the curve in Fig. 7 and the absence of the hysteresis confirm the superparamagnetic properties of the obtained SPIONs. The results ( Fig. 7) indicate that the nanoparticles are highly magnetic, with the saturation magnetization being equal to 123 ± 12 emu g⁻¹ Fe—very close to the value measured for magnetite (Jung and Jacobs 1995). Taking into account the total mass of the coated nanoparticles, the saturation magnetization reaches the value of 61 ± 6 emu g⁻¹, which is comparable to the ones measured for similar superparamagnetic nanoparticles (Matsumoto and Jasanoff 2008). Magnetic susceptibility of SPION1 was also determined in 295–323 K temperature range. The real part of susceptibility was found to be ca. 1.4 cm³ g⁻¹ and slightly decreasing for higher temperatures. Positive value of this parameter is characteristic for paramagnetic material. The FC/ZFC (field cooling/zero field cooling) measurements were also performed for dried SPION1 (see Electronic Supplementary Materials). However, the obtained results suggest further aggregation of SPION1 after drying and cannot be directly compared to the particles’ diameter measured for nanoparticle suspension (see Fig. 3).

In order to determine relaxivity r ₁ and r ₂ for SPIONs, the following relations were used: where R ₁ (R ₂) is the longitudinal (transverse) relaxation rate; T ₁ (T ₂) is the longitudinal (transverse) relaxation time; R ₁ ⁰ (R ₂ ⁰ ) is the longitudinal (transverse) relaxation rate in the absence of SPIONs; r ₁ (r ₂) is the longitudinal (transverse) relaxivity; and c _Fe is the iron concentration in the samples.

Values of relaxivity, r ₁ and r ₂, indicate whether SPIONs could be effective contrast agents for MRI. The iron oxide nanoparticles are contrast agents affecting T ₂; so for the best performance, r ₂ values should be very high, while T ₁ values are only weakly dependent on the contrast concentration implying low r ₁ values. As presented in Fig. 8 and Table 3, the fabricated SPIONs are characterized by relatively high r ₂ values that are also comparable to the values determined under the same measurement conditions for the commercial contrast agent, FeREX. It should be noticed that for negatively charged nanoparticles, SPION1/ACh, r ₂ was determined to be especially high and equal to 369 ± 3 mM⁻¹ s⁻¹ which is larger by more than 2.5 times than the value for FeREX. These materials seem to be very attractive for MRI applications, considering also that other commercial contrast agents like Sinerem/Combidex or Endorem/Feridex have r ₂ values equal to 65 and 120 mM⁻¹ s⁻¹, respectively (both determined at 1.5 T) (Laurent et al. 2008).

The observed relatively high relaxivity values for the obtained nanoparticles may be explained in terms of enhancement effect of the highly hydrated coating that has been only recently postulated (de Haan and Paquet 2011). For the highly charged polymeric coatings applied in this study, one can expect high hydration, definitely much higher than that functioning for uncharged or even hydrophobic coatings. It has to be stressed that water diffusion in the presence of the particles with the hydrophilic polymer shells may be hampered, what has been proven by observation of decrease of the water diffusion coefficients measured for that kind of polymers (Kwak and Lafleur 2003). This is evidenced here by significant increase of r ₂ value (see Table 3) only after deposition of ACh on SPION1 without any change in the inorganic part of the nanoparticles and with only small variation in the average size of the aggregates.