English

• Iron oxide nanoparticles (IONPs) are considered to be nontoxic, biodegradable, biocompatible and efficiently cleared from the human body via the pathways of iron metabolism [1, 2]. Moreover, IONPs possess the ability to enhance magnetic resonance imaging (MRI) contrast, as well as absorb the energy of an oscillating magnetic field and convert it into heat [3, 4]. These factors together with their potential for tailored modification (in terms of size, surface properties, and so on) make IONPs-based agents well suited for theranostic applications, and some of them have already been approved for clinical use in magnetic resonance imaging (MRI) diagnosis and hyperthermia [4, 5]. Recently, increasing efforts have been devoted to the design and fabrication of IONPs-based system for targeted-specific therapy which was focused on achieving active targeted delivery and environment responsive release of therapeutic drugs [6-12]. All these applications require surface modification of the IONPs, which not only function to improve the colloidal stability and biocompatibility, but may also provide chemical handles for conjugation of drug and targeting molecules [13]. The nature of the surface coatings can affect important properties of IONPs, it is therefore critical for further understanding how these materials react to physiological conditions, which is still needed to fully exploit the potential of IONPs as theranostic applications [14].

  Chitosan has been considered one of the most promising biopolymers for biomedical and pharmaceutical applications due to its biodegradability, antimicrobial, non-toxicity, and anti-tumor properties [15]. Folic acid is a well-known targeting molecule possessing high affinity for the folate receptor (FR) which was limitedly distributed in normal tissues while upregulated on both cancer cells (primarily FR-α isoform) and activated macrophages (FR-β isoform) [16].

  In the present study, we prepared IONPs with different surface modifications with citric acid (CA), chitosan (CS) and folic acid conjugated chitosan (FA-g-CS), and then we investigated effects of these surface modifications on the physicochemical properties of IONPs. The anticancer drug doxorubicin was used as a model drug to investigate the drug loading capacity and release patterns of IONPs. Finally, in vitro cytotoxicity of unloaded and DOX loaded IONPs were studied on L929 (FRs negative mouse fibroblast cell line) and MDA-MB-231(FRs positive human breast cancer cell line) [8]. Citric acid coated iron oxide nanoparticle (CA-IONP) was synthesized with a modified chemical co-precipitation method [17] using citric acid as a stabilizer. Folic acid was conjugated to chitosan through EDC/NHS coupling to obtain the FA-g-CS. Chitosan or FA-g-CS modified IONP (CS-IONP or FA-g-CS-IONP) was prepared via electrostatic attraction of surface negatively charged CA-IONP to positively charged chitosan or FA-g-CS. CA-IONP, CS-IONP and FA-g-CS-IONP were well characterized using Fourier transform infrared spectrometer (FT-IR), powder Xray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta-potential, thermogravimetric analysis (TGA) and vibrating sample magnetometer (VSM). The anticancer drug loading was achieved via non-covalent bonding between IONPs and DOX.

  The size and morphology of CA-IONP, CS-IONP and FA-g-CSIONP were studied by TEM. CA-IONP (Fig. 1a) presented good dispersity in water, and a broad size distribution spanned from 4 nm to 20 nm. CS-IONP (Fig. 1b) and FA-g-CS-IONP (Fig. 1c) revealed the encapsulation of IONPs in clusters with sizes around 200 nm. The formation of such aggregated clusters might be due to the protonation of amine groups all along the polymer chains (chitosan and FA-g-CS) in the acidic reaction medium during the surface modification step, which was linked to carboxylic groups in multiple particles [18].

  Figure 1

  

  Figure 1.  TEM images of (a) CA-IONP, (b) CS-IONP and (c) FA-g-CS-IONP. The embedded scale bars correspond to 20 nm

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  Surface modifications of IONPs were investigated by FT-IR spectroscopy. FT-IR analysis of CA-IONP (Fig. 2a) showed bands corresponding to carboxylate (COO^-) stretching (1629 cm^-1 and 1400 cm^-1) [19], confirming the presence of citric acid layers. CS-IONP (Fig. 2a) displayed bands corresponding to the O-H and N-H (primary amine) stretching vibrations (3405 cm^-1), the aliphatic C-H stretching vibration (2921 cm^-1) and the N-H bending vibration of primary amine (1594 cm^-1), confirming the presence of chitosan layers. FT-IR analysis of FA-g-CS (Fig. S1 in Supporting information) exhibited bands corresponding to the C=O stretching vibration of secondary amide (1659 cm^-1), the O-H and N-H (primary amine) stretching vibrations (3409 cm^-1), the N-H bending vibration of primary amine (1620 cm^-1), the C-N-H bending vibration of amide group (1559 cm^-1) indicating the successful conjugation of folic acid to chitosan. FA-g-CS-IONP (Fig. 2a) showed bands corresponding to the O-H and N-H (primary amine) stretching vibrations (3382 cm^-1), the aliphatic C-H stretching vibration (2925 cm^-1) and the C=O stretching vibration of secondary amide (broad band near 1603 cm^-1), confirming the presence of FA-g-CS layers.

  Figure 2

  

  Figure 2.  Physicochemical properties of CA-IONP, CS-IONP and FA-g-CS-IONP. (a) FT-IR spectra, (b) powder XRD patterns, (c) TGA curves and (d) hysteresis loops

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  The crystalline structure of CA-IONP, CS-IONP and FA-g-CSIONP was characterized by powder XRD analysis. The XRD pattern of CA-IONP (Fig. 2b) contained the peaks at 2θ values 30.1°, 35.5°, 43°, 53.9°, 57.6° and 62.8° corresponding to the (220), (311), (400), (422), (511) and (440) planes of magnetite, the values were compliant with those reported earlier [20]. After modified with chitosan or FA-g-CS, the same characteristic peaks were exhibited, indicating that CS-IONP and FA-g-CS-IONP retained the crystalline structure of magnetite.

  The mass ratios of organic layers in the CA-IONP, CS-IONP and FA-g-CS-IONP were estimated by thermogravimetric analysis (TGA). Bare iron oxide nanoparticles, pure chitosan and FA-g-CS were used as controls to determine the non-combustible residues. As shown in the thermogravimetric curves (Fig. 2c), the percentage mass loss under 200 ℃ was 6% for CA-IONP and CS-IONP, and 7% for FA-g-CS-IONP, which was assigned to the reduction of physically adsorbed water. The percentage mass loss between 200–900 ℃ was 10% for CA-IONP, 25% for CS-IONP and 14% for FA-g-CS-IONP, which was due to the thermal decomposition of organic layers. Mass ratio of non-combustible residues was 96%, 30% and 26% for IONP, chitosan and FA-g-CS, respectively (Fig. S2 in Supporting information). Based on the TGA curves, the mass ratio of organic layers was calculated to be 6.5%, 32% and 15% for CA-IONP, CS-IONP and FA-g-CS-IONP, respectively.

  The magnetic properties of IONPs were investigated using a vibrating sample magnetometer (VSM) in a magnetic field ranged of -5000 Oe to 5000 Oe at 300 K. The saturation magnetization at maximum field was normalized to the gram of particles. The hysteresis loops (Fig. 2d) presented no hysteresis for all IONPs, confirming that all of them possessed the superparamagnetic behavior; only a negligible value (coercivity less than 10 Oe) was observable, which was attributed to the remanent field of the VSM coils. The saturation magnetization was 47.39 emu/g, 40.5 emu/g and 43.8 emu/g for CA-IONP, CS-IONP and FA-g-CS-IONP, respectively (Fig. 2d). The decrease in saturation magnetization after surface modified with chitosan or FA-g-CS might be attributed to the increased amount of the non-magnetic organic layers onto the nanoparticles, this finding was validated by the excellent agreement between the saturation magnetization values measured via VSM and the mass ratio of organic layers calculated via TGA [21].

  Zeta potentials were recorded for IONPs. CA-IONP was shown to possess a negatively charged surface with a zeta potential value of -35.4 ± 2.9 mV (Table 1), confirming the presence of anionic citric acid on the surface of nanoparticles. After the treatment with cationic chitosan, the obtained CS-IONP exhibited a positively charged surface with a zeta potential value of 33.9 ± 0.8 mV (Table 1), confirming the successful surface modification of particles with cationic chitosan. Compared with CS-IONP, FA-gCS-IONP showed a lower surface potential with value of 19.4 ± 0.3 (Table 1), which was due to the reduced amino groups in FA-g-CS, indicating the presence of FA-g-CS coatings on the FA-g-CS-IONP.

  Table 1

  

  Table 1.  Summary of hydrodynamic diameter and surface potential of IONPs based on dynamic light scattering and zeta potential analyses

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  The hydrodynamic diameter of IONPs and their dispersion state in solutions were estimated by dynamic light scattering (DLS). CA-IONP was well dispersed in DI water and 0.01 mol/L buffers with pH ranging from 4.6 to 6.4, and exhibited small hydrodynamic diameter spanned from 28 nm to 42 nm (Table 1). However, CA-IONP highly agglomerated in 0.01 mol/L buffer with pH 7.3, which exhibited a large hydrodynamic diameter above 1000 nm (Table 1). CS-IONP and FA-g-CS-IONP presented good dispersity in DI water and 0.01 mol/L buffers with pH 4.6 and 5.6, and showed hydrodynamic diameters bellow 350 nm (Table 1). However, CS-IONP and FA-g-CS-IONP quickly formed large agglomerates in 0.01 M buffers with higher pH (6.4 and 7.3), which exhibited a large hydrodynamic diameter above 1000 nm (Table 1).

  Zeta potentials were also recorded for DOX loaded IONPs. DOX@CA-IONP exhibited a negatively charged surface with an absolute potential value of around 6 mV (Table 1) which was lower than that of CA-IONP, indicating the loading of DOX on the CA-IONP, as it was done mainly via electrostatic attraction of positively charged DOX and negatively charged surface of CA-IONP. DOX@CS-IONP and DOX@FA-g-CS-IONP showed positively charged surface with absolute potential values of 66 mV and 60 mV (Table 1), respectively, which was higher than those of CS-IONP and FA-g-CS-IONP, indicating the loading of DOX on the CS-IONP and FA-g-CS-IONP. The obtained DOX@CA-IONP was highly agglomerated (Table 1), which was due to the decreased electrostatic repulsion that was no longer enough to guarantee their colloidal stability. DOX@CS-IONP and DOX@FA-g-CS-IONP were found to be well dispersed in DI water and 0.01 mol/L buffers with pH 4.6 and 5.6, however, they quickly agglomerated in 0.01 mol/L buffers with pH 6.4 and 7.3 (Table 1).

  As it is well known, the stability of nanoparticles wearing surface charges was mainly ensured by electrostatic repulsions between grains. Surface charges of nanoparticles were often introduced via surfactants bearing -COO^- or -NH³⁺ and their protonation state determined the net surface charge. The protonation state of carboxyl or amino groups depend on the surrounding pH. Ions of surroundings will shield the surface charges of nanoparticles [22]. Hence, the pH and ionic strength of surroundings will change the electrostatic interactions between particles, they can therefore significantly influence the dispersion state of particles [22, 23]. Surface negatively charged nanoparticles have been reported to agglomerate at pH 7 after an addition of electrolyte, which increases the ionic strength, decreases the repulsions and induce the agglomeration of nanoparticles [22], this might as well explain the agglomeration of CA-IONP in phosphate buffer (0.01 mol/L, pH 7.3) (Table 1). Positively charged amino groups of chitosan and FA-g-CS tended to deprotonate when surrounding pH increased, which resulted in the reduction of charge density. It was the deprotonation of amino groups and the 'charge shielding' phenomenon in solutions with high ionic strength that induced the poor stability of CS-IONP and FA-gCS-IONP in 0.01 mol/L buffers with pH of 6.4 and 7.3.

  The loadings of DOX in the DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP was confirmed by FT-IR analyses. The FT-IR spectrum of doxorubicin (Fig. 3a) showed bands corresponding to aliphatic C-H stretching vibration (2935 cm^-1), N-H bending vibration of primary amine (1617 cm^-1), skeletal vibration of aromatic ring (1414 cm^-1), C-O stretching vibration (1073 cm^-1), C-H out-of-plane vibration in the aromatic ring (846 cm^-1). Moreover, these bands were also presented in the FT-IR spectra of DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP (Fig. 3a). For DOX@CA-IONP, these bands shifted to 2931 cm^-1 (aliphatic C-H stretching vibration), 1620 cm^-1 (N-H bending vibration of primary amine), 1387 cm^-1 (skeletal vibration of aromatic ring), 1075 cm^-1 (C-O stretching vibration) and 843 cm^-1 (C-H out-ofplane vibration in the aromatic ring). For DOX@CS-IONP, these band shifted to 2925 cm^-1 (aliphatic C-H stretching vibration), 1613 cm^-1 (N-H bending vibration of primary amine), 1385 cm^-1 (skeletal vibration of aromatic ring), 1073 cm^-1 (C-O stretching vibration) and 850 cm^-1 (C-H out-of-plane vibration in the aromatic ring). For DOX@FA-g-CS-IONP, these bands shifted to 2925 cm^-1 (aliphatic C-H stretching vibration), 1605 cm^-1 (N-H bending vibration of primary amine), 1382 cm^-1 (skeletal vibration of aromatic ring), 1069 cm^-1 (C-O stretching vibration) and 849 cm^-1 (C-H out-of-plane vibration in the aromatic ring). These results confirmed that DOX was successfully loaded onto the nanoparticles.

  Figure 3

  

  Figure 3.  (a) FT-IR spectra of DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP; (b) DOX release patterns in 0.01 mol/L phosphate buffer at pH 7.3 and 37 ℃; cell viability of (c) L929 and (d) MDA-MB-231 after incubation with IONPs for 72 h

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  DOX encapsulation efficiency was expressed as the weight ratio of loaded DOX to total DOX used for encapsulation. Encapsulation efficiency was 31%, 13% and 14% for DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP, respectively. DOX loading capacity was expressed as the weight ratio of loaded DOX to iron contents of NPs. DOX loading capacity was 13 wt%, 6 wt% and 7 wt% for DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP, respectively. The high DOX loading capacity of DOX@CA-IONP was attributed to the strong attraction between negatively charged CA-IONP with positively charged DOX. However, the presence of positively charged chitosan and FA-g-CS undermined the electrostatic attraction between DOX and CA-IONP, which resulted in a lower DOX loading. The slightly higher DOX loading of DOX@FA-gCS-IONP compared with DOX @CS-IONP might be due to the anionic carboxylic groups of folic acid ligands.

  Drug release behaviors from DOX loaded IONPs were investigated in 0.01 mol/L buffer with pH of 7.3 at physiological temperature of 37 ℃. Aliquots of the release medium were withdrawn at predetermined time intervals, and the amount of DOX in the medium was estimated by measuring their absorbance at 490 nm and converting to the concentration of DOX via the DOX calibration curves (Fig. S3 in Supporting information). In all cases, DOX was gradually released from the drug loaded IONPs, and the cumulative release amount after 96 h was 24%, 16% and 17% for DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP, respectively. For DOX@CA-IONP, its drug release rate was faster than those of DOX@CS-IONP and DOX@FA-g-CS (Fig. 3b). The loading of DOX on CA-IONP was done via electrostatic attraction and DOX was almost localized on the particles surface. For DOX@CS-IONP and DOX@FA-g-CS-IONP, DOX loading was achieved via multiple forces and DOX was mainly localized at the inner layer while biopolymers composed the out layers of the organic coatings. DOX localized at the inner layer usually possesses a lower diffusion rate than surface bound DOX, this might explain the slower drug release from DOX@CS-IONP and DOX@FA-g-CS-IONP when compared with DOX@CA-IONP.

  In vitro cytotoxicity of unloaded and DOX loaded IONPs was evaluated by determining the viability of cells after incubation with medium containing the respective nanoparticles for 72 h with the MTT assay. MDA-MB-231 cells and L929 cells were used for cytotoxicity testing. Iron concentrations of CA-IONP, CS-IONP and FA-g-CS-IONP dispersions were calculated via the iron calibration curve (Fig. S4 in Supporting information). The viability of both cell lines after incubation with unloaded nanoparticles was nearly unchanged with the increase in iron concentration in the range of 10 μg/mL to 40 μg/mL (Figs. 3c and d). After treatment with unloaded nanoparticles at an iron concentration of 40 μg/mL, the viability of L929 vs. that of MDA-MB-231 was 49% vs. 60%, 83% vs. 90% and 77% vs. 87% for CA-IONP, CS-IONP and FA-g-CS-IONP, respectively. Results indicated that CA-IONP possessed significant cytotoxicity on both cell lines, moreover, the presence of chitosan and FA-g-CS layers remarkably reduced the cytotoxicity of IONPs. After incubation with DOX loaded nanoparticles, the viability of MDA-MB-231 cells significantly decreased with the increase in iron concentration, this was due to the simultaneous increase in anticancer drug. Moreover, DOX@FA-g-CS-IONP exhibited significant cytotoxicity on MDA-MB-231 cells while showing nearly no cytotoxicity on L929 cells, which might be attributed to the presence of folic acid, which was a tumor-specific affinity ligand, on the particles. After treatment with DOX loaded nanoparticles at an iron concentration of 40 μg/mL, the viability of L929 vs. that of MDA-MB-231 was 45% vs. 27%, 86% vs. 67% and 76% vs. 35% for DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP, respectively (Figs. 3c and d). Among these DOX loaded IONPs, DOX@CAIONP possessed the highest cytotoxicity, which was achieved by their relatively high drug loading capacity as well as their relatively fast drug release rate when compared with DOX@CS-IONP and DOX@FA-g-CS-IONP.

  In conclusion, we successfully prepared IONPs modified with citric acid, chitosan and folic acid conjugated chitosan for their surfaces, respectively. CA-IONP dispersion was composed of monocrystalline particles while CS-IONP and FA-g-CS-IONP were composed of polycrystalline aggregates, which was due to the bonding of positively charged high molecular chitosan and FA-g-CS to carboxylic groups in multiple particles. CA-IONP, CS-IONP and FA-g-CS-IONP all retained the crystalline structure of magnetite and the superparamagnetic behavior. However, the saturation magnetization of IONPs decreased with the increase in the amount of their organic coatings. Unloaded and DOX loaded IONPs, except DOX@CA-IONP, exhibited excellent stability in DI water. The pH and ionic strength of surroundings significantly influenced the colloidal stability of surface charged IONPs via changing the electrostatic interactions between particles. DOX@CA-IONP possessed the highest DOX loading due to the strongly electrostatic attractions between CA-IONP and DOX, however, they were highly agglomerated due to the reduction of surface net charge and therefore, the electrostatic repulsions between particles were no longer enough to ensure their colloidal stability. DOX@CS-IONP and DOX@FA-g-CS-IONP showed comparable drug loading capacity and good dispersity in water. DOX release rate of IONPs was slowed down in the presence of chitosan or FA-g-CS coatings, which was due to the protection of polysaccharide out layer. CA-IONP possessed significant cytotoxicity on both cell lines, however, the presence of chitosan and FA-g-CS layers remarkably reduced the cytotoxicity of IONPs. Moreover, DOX@FA-g-CS-IONP showed significant cytotoxicity on FRs positive breast cancer cells while exhibiting nearly no cytotoxicity on FRs negative normal cells, this might be attribute to the presence of folic acid which was a tumor-specific affinity ligand on the particles.

  Although nanotechnology have been widely applied in bioassay [24-29], and magnetic nanoparticles have also found many applications [30-38], especial in biomedical fields [39-48], the results presented in this study were valuable to the design and fabrication of IONPs-based system for better theranostic applications.

  Acknowledgments

  This work was supported by the State Key Basic Research Program of the PRC (No. 2014CB744501), the National Key Research and Development Program of China (No. 2017YFA0205301), the National Natural Science Foundation of China (Nos. 61527806, 61471168 and 61871180) and Open Funding of State Key Laboratory of Oral Diseases (No. SKLOD2018OF02).

  Appendix A. Supplementary data

  Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.10.038.

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  Figure 1  TEM images of (a) CA-IONP, (b) CS-IONP and (c) FA-g-CS-IONP. The embedded scale bars correspond to 20 nm

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  Figure 2  Physicochemical properties of CA-IONP, CS-IONP and FA-g-CS-IONP. (a) FT-IR spectra, (b) powder XRD patterns, (c) TGA curves and (d) hysteresis loops

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  Figure 3  (a) FT-IR spectra of DOX@CA-IONP, DOX@CS-IONP and DOX@FA-g-CS-IONP; (b) DOX release patterns in 0.01 mol/L phosphate buffer at pH 7.3 and 37 ℃; cell viability of (c) L929 and (d) MDA-MB-231 after incubation with IONPs for 72 h

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  Table 1.  Summary of hydrodynamic diameter and surface potential of IONPs based on dynamic light scattering and zeta potential analyses

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