Fe3O4 magnetic nanoparticles for application in liver magnetic resonance imaging

Journal of Magnetism and Magnetic Materials 388 (2015) 116–122

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Preparation and characterization of biofunctionalized chitosan/Fe3O4 magnetic nanoparticles for application in liver magnetic resonance imaging Xiaoli Song n, Xiadan Luo, Qingqing Zhang, Aiping Zhu n, Lijun Ji, Caifeng Yan College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 17 February 2014 Received in revised form 12 May 2014 Accepted 5 April 2015 Available online 7 April 2015

Biofunctionalized chitosan@Fe3O4 nanoparticles are synthesized by combining Fe3O4 and CS chemically modified with PEG and lactobionic acid in one step. The biofunctionalized nanoparticles are characterized by TEM, X-ray, DLS, zeta-potential and magnetic measurements. The in vitro and in vivo behaviors of the biofunctionalized nanoparticles, especially, the cytotoxicity, the protein resistance, metabolism and iron toxicity are assessed. The functional groups, PEG enable the nanoparticles more biocompatible and the lactobionic acid groups enable liver targeting. The potential applications of the nanoparticles in liver magnetic resonance imaging are confirmed. The results demonstrated that the nanoparticles are suspension stability, non-cytotoxicity, non-tissue toxicity and sensitive in liver magnetic resonance imaging, representing potential tools for applications in the biomedical field. & 2015 Elsevier B.V. All rights reserved.

Keywords: PEG/lactobionic acid-chitosan Magnetism Nanoparticles Liver targeting Magnetic resonance imaging

1. Introduction During last decade, nanomaterials have been the focus of an intensive research due to the functionalities unavailable to bulk materials. Especially, biochemically functionalized nanoparticles can be used in many different biomedical applications, such as targeted drug delivery [1], bio-separation [2], hyperthermia, and magnetic resonance imaging (MRI) [3, 4]. For MRI, magnetite (Fe3O4) attracted considerable interest due to their chemical stability, magnetic properties, and biocompatibility [5, 6]. Ideal magnetite for MRI should fulfill some necessary properties, such as superparamagnetism, strong magnetic responsiveness, high stability, narrow size distribution, and biocompatibility. Magnetic nanoparticles possess a good performance of the properties above except dispersion stability. Thus, surfactants are usually used to coat magnetic nanoparticles to prevent aggregation caused by magnetic dipole–dipole attractions between particles [7, 8]. Functionality of magnetic nanoparticles is largely dependent on the properties of their surface coatings, which can change the surface charge of the particles, reduce the risk of immunogenicity, and increase the biocompatibility and the cellular uptake. Among the coating materials, chitosan (CS) has drawn considerable attention due to its excellent properties, such as nontoxicity, hydrophily, biocompatibility, biodegradability [9–11] and n

Corresponding author. Fax: 86 514 87975244. E-mail address: [email protected] (X. Song).

http://dx.doi.org/10.1016/j.jmmm.2015.04.017 0304-8853/& 2015 Elsevier B.V. All rights reserved.

with many reactive functional groups [12] that can serve as an anchor to conjugate targeting ligands, therapeutics and imaging agents. However, CS is only soluble in aqueous medium in the presence of acid, which limits its application in some biomedical area [12, 13]. Polyethylene glycol (PEG) is a neutral polymer with a number of outstanding physicochemical and biological properties, including non-toxicity, water solubility, protein resistance and immunogenicity. It is usually the hydrophilic structure chosen to provide a hydrated steric barrier for materials [14]. The PEG outer shell can also reduce the platelet deposition on the surfaces of the materials and prolong the circulation time in vivo [15]. To date, PEG is one of only a small number of synthetic polymers approved by the FDA [16] and has been used in a wide range of biomedical applications [17]. Lactobionic acid (LA) is an endogenous substance present in the human body. The bearing of galactose unit in LA acts as a specific targeting ligand for asialoglycoprotein receptor (ASGPR) on hepatocytes [18–20]. Above all, the coating of Fe3O4 nanoparticles surface with CS chemically modified with PEG and LA can further improve its dispersion stability, biocompatibility, circulation time and especially the targeting for hepatocytes. Several methods are commonly used for the preparation of magnetic polymer particles, such as miniemulsion polymerization in the presence of iron oxide [21], emulsion polymerization [22, 23], dispersion polymerization [24], suspension polymerization [25] and encapsulation [26]. Among these methods, direct encapsulation of magnetic cores with a polymer is the most simple and green approach since no other agents, such as initiator,

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emulsifier is introduced. Therefore, the objectives of the present work were: first to modify the CS with PEG and LA to obtain a water-soluble, biocompatible, self-assembly and liver targeting CS derivative (PEG/ LA-CS), second to encapsulate the Fe3O4 nanoparticles by the selfassembly of PEG/LA-CS, third to evaluate the in vitro and in vivo behavior of the PEG/LA-CS@Fe3O4 nanoparticles. More specifically, the cytotoxicity and the protein resistance of the nanoparticles were evaluated. Finally, the potential application in liver MRI was confirmed.

2. Materials and methods 2.1. Materials Ferric chloride hexahydrate (FeCl3
6H2O), ferrous chloride tetrahydrate (FeCl2
4H2O), ammonium hydroxide (NH4OH), dimethyl sulfoxide (DMSO), and succinic anhydride were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Chitosan (CS, 500KD, 90% deacetylated) was obtained from Lianyungang Biologicals Inc. (Jiangsu, China). Methoxy poly (ethylene glycol) (mPEG, 2000), HCl, Pyridine, N-hexane, Benzene, Tetramethylethylenediamine (TEMED) and BSA were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. EDC.HCl and NHS was obtained from J&K Scientific Ltd. The deionized water (ddH2O) was deoxygenated by bubbling with N2 gas for 1 h prior to use. All the other reagents were of analytical grade, and used without further purification.

Fig. 1. FT-IR spectrum of PEG/LA-CS@Fe3O4 NPs.

prepared Fe3O4 NPs was dispersed in 25 mL of 0.4 mg/ml PEG/LACS (pH 7.4), and mechanical stirred (500 r/min) for 12 h at room temperature under N2 protection to ensure the even coating of the Fe3O4 NPs. Then the unbound PEG/LA-CS was removed by several centrifugation-redispersion cycles. The PEG/LA-CS@Fe3O4 NPs were finally obtained by freeze-drying. 2.5. Characterization of PEG/LA-CS@Fe3O4 NPs

2.2. PEG/LA-CS synthesis To synthesize carboxy terminated Poly (ethylene glycol) methyl ether (mPEG-COOH), Poly (ethylene glycol) monomethyl ether (mPEG) (Mw 2000) (5 g, 2.5 mmol) was reacted with succinic anhydride (0.3 g, 3 mmol) with a catalytic amount of pyridine at 65 ◦C for 24 h. The mixture obtained was reprecipitated in diethyl ether. Then the precipitate was collected, washed with diethyl ether once again, and dried in vacuum to obtain mPEG-COOH. To synthesize PEG/LA-CS, 1 g CS was dissolved in 6 mL acetic acid (2%), diluted into 20 mL TEMED/HCl buffer solution (pH 4.7) and stirred for 30 min. Then 3.83 mg EDC and 2.30 mg NHS was added to the solution above respectively. After that, 0.16 mg mPEG-COOH and 1.43 mg LA was added and mechanical stirred for 72 h under 35 °C at the pH controlled between 4 and 6. The crude products were dialyzed against ddH2O for 4 days and PEG/LA-CS was finally obtained by freeze-drying.

A FT-IR spectrometer (IFS66/S) was used to characterize the modification of PEG/LA-CS onto the surface of Fe3O4 NPs. The FT-IR spectra were collected in the frequency range of 4000–500 cm  1 at a resolution of 4 cm  1. All spectra were normalized against a background of an air spectrum and recorded as absorbance values at each data point in triplicate. The crystal structure of the PEG/LA-CS@Fe3O4 NPs was examined on an XD-3A power diffractometer using a monochromatized X-ray beam with nickel-filtered Cu Kα radiation in the range of 5–80° (2θ) at 40 kV and 30 mA. The size and size distribution of the PEG/LA-CS@Fe3O4 NPs were measured on a dynamic light scattering (DLS) system

2.3. Preparation of Fe3O4 nanoparticles Fe3O4 nanoparticles (Fe3O4 NPs) were prepared by traditional chemical precipitation method [6] from aqueous iron salt solutions by means of alkaline media. Briefly, 5 mL of iron solution containing 0.1 M Fe2 þ and 0.2 M Fe3 þ was added slowly to 50 mL of NH4OH (29.4 wt%) solution under N2 protection and magnetic stirring vigorously for 30 min at room temperature. After stopping stirring, a strong magnet was used to settle down the black precipitate, and the powder was washed with the freshly prepared and deoxygenated ddH2O for five times to remove the excess ammonia. The black Fe3O4 NPs was finally obtained by freezedrying. 2.4. Preparation of PEG/LA-CS@Fe3O4 NPs The coat of PEG/LA-CS on Fe3O4 NPs was carried out in one-step through the self-assembly of PEG/LA-CS. Briefly, 200 mg as-

Fig. 2. The size of PEG/LA-CS@Fe3O4 NPs measured by DLS.

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(DLS-5022F). All the measurements were performed at room temperature and the scattering angle was set to 90°. The mean NPs diameter was calculated using differential size distribution processor intensity analysis program. The size distribution of the NPs was characterized statistically by polydispersity index (PDI). For the measurements of zeta potential, PEG/LA-CS@Fe3O4 NPs were suspended in ddH2O filtered by films with pore size of 0.22 mm, and ζ-potential was measured on a Malvern ZetaSIZER Nanoseries ZS (Malven Instruments, woecestershire, UK) in triplicate. The morphology of the PEG/LA-CS@Fe3O4 NPs was observed by a transmission electron microscope (TEM) (TECHAI-12). For sample preparation, a drop of sample solution (1 mg/mL in ddH2O) was placed on a 300-mesh copper grid coated with carbon. Subsequently, the sample was dried and negatively stained by a 2% (w/ w) uranyl acetate solution. The magnetic properties of the PEG/LA-CS@Fe3O4 NPs (100 mg) were measured on a BHV-5 vibrating sample magnetometer in the range of  6000 to 6000 Oe at room temperature. 2.6. Protein resistance by electrochemical impedance spectroscopy (EIS) 2.6.1. Electrode modification 5 μL PEG/LA-CS@Fe3O4 NPs (2 mg/mL) solution was dropped on the surface of the pretreated glassy carbon electrode. Then the electrode was dried in a refrigerator at 4 °C. Finally, the electrode was rinsed in ddH2O to remove the NPs adsorbed on the surface of the electrode. The modified electrode above were immersed in a 10 mg/mL BSA solution and placed in a water bath of 37 °C for 30 min, and then rinsed with ddH2O to remove the excess BSA. Finally, the prepared electrodes were immersed in a phosphate buffer solution (pH¼7.4) and stored at 4 °C until further use. 2.6.2. Electrochemical impedance spectroscopy (EIS) An electrochemical cell consisting of three electrode systems: the glassy carbon electrode or the modified electrode as working electrode, the saturated calomel electrode as reference electrode, and the platinum electrode as counter of electrodes was used to measure the EIS on an autolab electrochemical workstation (Ecocoemie, Netherlands). The ESI was measured in the range of 10  1– 106 Hz. The amplitude of the sinusoidal potential was set to 10 mV using open-circuit potential. The solution containing 1 mM Fe (CN)63  /4  , 0.1 M phosphate and 0.1 M KNO3 was used as the

tested buffer solution. 2.7. Cell proliferation assay of PEG/LA-CS@Fe3O4 NPs K562 cells were used for the analysis of cytotoxicity in vitro. The cells (5  103 cells/mL) were placed into 96-well tissue-culture plates and incubated at 37 °C with CO2. After 24 h, cells were treated with PEG/LA-CS@Fe3O4 NPs at different concentrations. Untreated cells were used as controls. Plates were incubated in a humidified 5% CO2 balanced-air incubator at 37 °C for different times. Then 20 μL of 5 mg/mL MTT solution was added to each well and the plates were incubated for another 4 h. After that, 50 μL DMSO was added to each well. The results were read on ELISA plate reader (A5002, Tecan, Austria), using a wavelength of 490 nm. 2.8. In vitro and in vivo MRI of PEG/LA-CS@Fe3O4 NPs 2.8.1. In vitro MRI 1.5 mLof PEG/LA-CS@Fe3O4 NPs suspension (PBS, pH 7.4) was installed in three EP tubes, respectively. The tubes were fixed vertically on the plastic board, standing for one month and then MRI was carried out on an AW 4.2 working station. The signal on different levels of the tubes was recorded. 2.8.2. In vivo liver MRI For in vivo MRI, Ten male SD rats of 3–5 months (Central Animal Services, Yangzhou University) were intraperitoneal anesthetized (40 mg/kg) with 2% sodium pentobarbital and then were intravenously injected of PEG/LA-CS@Fe3O4 NPs PBS suspension through the tail vein (0.05 mmol/kg). After recovering for 30 min, the rates were placed in the knee coil of a signal Echospeed 1.5 T scanner. MRI through the PDWI of the liver was obtained using a fast spin-echo sequence with a spin echo time of 10 ms. Image slices were 2–4 mm thick and 0.5–1.0 mm spaced. A 256  224 image reconstruction matrix was used over a field of view of 12 cm. 2.9. Metabolism and iron toxicity essays In order to detect the metabolism and iron toxicity of the PEG/ LA-CS@Fe3O4 NPs, 0.5 mmol/kg of PEG/LA-CS@Fe3O4 NPs PBS suspension was injected through the tail vein of the SD rats. MRI was carried out after 24 h or 7 days as above, respectively. Prussian blue staining analysis of the liver of SD rats was also carried out

Fig. 3. TEM morphologies of Fe3O4 NPs (a) and PEG/LA-CS@Fe3O4 NPs (b).

X. Song et al. / Journal of Magnetism and Magnetic Materials 388 (2015) 116–122

Fig. 4. X-ray diffraction patterns of unmodified Fe3O4 NPs (a) and PEG/LACS@Fe3O4 NPs (b).

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Fig. 7. The dependence of the PEG/LA-CS@Fe3O4 NPs concentration on the relative cell viability for 7 days of cell culture.

Fig. 5. Magnetization curve of Fe3O4 NPs and PEG/LA-CS@Fe3O4 NPs.

Fig. 8. The in vitro MRI of PEG/LA-CS@Fe3O4 NPs at sequence T2WI (a) and T1WI (b).

Table 1 The relative signal strength at different levels of PEG/LA@ Fe3O4 NPs tubes. levels

1

2

3

4

5

signal strength (T2WI/T1WI)

16.63/ 41.45

16.55/ 41.33

16.84/ 41.25

16.78/ 41.74

16.84/ 41.72

3. Results and discussion Fig. 6. EIS spectra of PEG/LA-CS@Fe3O4 NPs modified electrodes in solution containing 1 mM Fe (CN)63  /4  , 0.1 M phosphate and 0.1 M KNO3 before (a) and after being immersed in a 10 mg/mL BSA solution for 30 min (b).

after 24 h and 7 days to detect the PEG/LA-CS@Fe3O4 NPs. Finally, the pathological analysis of the liver tissue of SD rats was conducted after 7 days.

3.1. Synthesis and characterization of PEG/LA-CS@Fe3O4 NPs In this report, biocompatible and liver targeting CS derivative (PEG/LA-CS) was synthesized by coupling PEG and LA onto CS with EDC and NHS as the catalyst. Encapsulation Fe3O4 NPs into PEG/ LA-CS was prepared by the self-assembly under mechanical

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Fig. 9. PDWI images of rat liver before (a) and after (b) injection of PEG/LA-CS@Fe3O4 NPs Downward white arrow points the rat liver.

stirring with N2 protecting. To confirm the modification of PEG/LACS onto the surface of Fe3O4 NPs, the FT-IR spectra were collected. For PEG/LA-CS (Fig. 1), the characteristic peaks of amide bond appeared at 1642 and 1512 cm  1. The peak at 3439 cm  1 was assigned to O-H stretch overlapped with N–H stretch, the peak at 2896 cm  1 was assigned to C–H stretch and the peak at 1086 cm  1 was assigned to the C–O stretch. It can be seen from the FT-IR of Fe3O4 NPs, there was a characterized peak at 585 cm  1. In the FT-IR spectra of PEG/LA-CS@Fe3O4 NPs, all the characterized peaks of PEG/LA-CS and Fe3O4 NPs appeared in the corresponding wavenumbers, indicating the modification of PEG/ LA-CS onto the surface of Fe3O4 NPs. For MRI applications, the size of the magnetic is an important factor. Thus the size and distribution of the PEG/LA-CS@Fe3O4 NPs were first measured by DLS (Fig. 2). The mean particle radius of the NPs is 49.5 nm with a polydispersity index of 0.125, suggesting the particle size is uniform. These results also indicate that there is no obvious aggregation of the NPs. This might be due to that the zeta potential of PEG/LA-CS@Fe3O4 NPs is þ33.3 71.1 mV, which can prevent the reunion of the particles and improve its monodisperse. However, the mean particle size for unmodified Fe3O4 NPs is 750 nm, suggesting a very fast and strong aggregation. As known, the dispersion stability of ferrofluids composed of Fe3O4

NPs is affected not only by the particle size distribution, but also by the surface charges of the particles in solution. An effective way to stabilize ferrofluids is electrostatic repulsion, which can be achieved by immobilizing charges on the particle surface. Therefore, Fe3O4 NPs are usually grafted with nonmagnetic substances to form a stable ferrofluid. Many kinds of natural (e.g., proteins and polysaccharides), synthetic (e.g., polyelectrolytes), and non-ionic polymers [e.g., poly (vinyl alcohol)] have been used for particle coating [27, 28]. To confirm that PEG/LA-CS is effective to stabilize the Fe3O4 NPs suspension, the hydrodynamic diameter of PEG/LACS@Fe3O4 NPs was also monitored by DLS at 3, 7, 14 and 30 days, respectively. As a result, there was almost no change of the hydrodynamic diameter of the PEG/LA-CS@Fe3O4 NPs, indicating the good dispersion stability of the suspension. To confirm the morphology of the NPs, TEM image (Fig. 3) was observed. As shown in Fig. 3, the resulted PEG/LA-CS@Fe3O4 NPs are mostly spherical as the unmodified Fe3O4 NPs and the average particle diameter of PEG/LA-CS@Fe3O4 NPs only increases a little, from 14.1 73.5 nm to 14.8 7 1.5 nm with a narrower distribution. However, comparing with the unmodified Fe3O4 NPs, the dispersion of PEG/LA-CS@Fe3O4 NPs significantly improved. The crystalline structure of Fe3O4 NPs and PEG/LA-CS@Fe3O4 NPs was determined by XRD with the diffractogram (Fig. 4). There are six

Fig. 10. PDWI images of rat liver before (a), 24 h after (b) and 7 days after (c) overdose injection of PEG/LA-CS@Fe3O4 NPs Downward white arrow points the rat liver.

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diffraction peaks: (220), (311), (400), (422), (511) and (400), assigned to both the Fe3O4 and PEG/LA-CS@Fe3O4 NPs, consistent with the standard pattern of a crystalline magnetite with a spinel structure [29], which indicates that the crystalline structure of Fe3O4 NPs has no change before and after modification. This is just consistent with that the coatings of PEG/LA-CS on the surfaces of Fe3O4 NPs are thin, the spinel shape of PEG/LA-CS@Fe3O4 NPs is well maintained. For MRI, the magnetic properties of the contrast agent should be demanded. The magnetization of ferromagnetic Fe3O4 bulk material is very sensitive to the size of the sample. Superparamagnetism occurs when the particle is small enough that thermal fluctuation can overcome the magnetic anisotropy. The magnetization hysteresis loops of Fe3O4 and PEG/LA-CS@Fe3O4 NPs at 300 K are shown in Fig. 5. The saturation magnetization of the Fe3O4 NPs and PEG/LA-CS@Fe3O4 NPs is 67.4 and 65.5 emu/g, respectively. The little decrease of the saturation magnetization is due to the polymer coated on the surface of the Fe3O4 NPs. The hysteresis loop confirmed the superparamagnetism since there is no remanence effect, indicating PEG/LA-CS@Fe3O4 NPs are able to meet the requirements of MRI. 3.2. Biocompatibility of PEG/LA-CS@Fe3O4 NPs by EIS Biocompatibility of materials usually contains cytocompatibility and hemocompatibility and should be carefully examined before being used as a new biomaterial. The protein adsorption can usually reflect the hemocompatibility of the materials and the cell viability usually reflects the cytocompatibility. Then the protein adsorption of PEG/LA-CS@Fe3O4 NPs was detected by EIS. EIS contains semicircle and linear parts. The highfrequency part of the semicircle corresponds to the electron transfer process, while the low-frequency part of the straight line corresponds to the diffusion process. The semicircle diameter of EIS (SDEIS) reflects the electron transfer resistance of the electrode surface, which can be used to monitor the changes of the electrode surface. Therefore it was simply done to study protein adsorption of PEG/LA-CS@Fe3O4 NPs by monitoring the diameter changes of modified electrode before and after incubation in a BSA solution [30]. Fig. 6 shows the ESI spectra of PEG/LA-CS@Fe3O4 NPs surface modified electrodes in 1 mM Fe (CN)63  /4  , 0.1 M phosphate and 0.1 M KNO3 solution before and after immersed in a 10 mg/ml BSA solution. As shown in Fig. 6, the SDEIS of the bare glassy carbon electrode is small. After the coating of PEG/LA-CS@Fe3O4 NPs, the SDEIS shows increasing tendency. However, there is only a little increase of the SDEIS after being immersed in a 10 mg/ml BSA solution for 30 min. These results explain that PEG/LA-CS@Fe3O4 NPs almost do not adsorb proteins and show excellent hemocompatibility. To confirm the cytocompatibility of PEG/LA-CS@Fe3O4 NPs, the effect of PEG/LA-CS@Fe3O4 NPs on K562 cell viability was investigated. Fig. 7 shows the dependence of the concentration of PEG/LA-CS@Fe3O4 NPs on the relative cell viability after 7 days incubation. As shown in Fig. 7, in the range of 0–0.5 mg/mL of PEG/ LA-CS@Fe3O4 NPs, the relative cell viability just decreased a little. Compared with the control, the cell viability still maintained more than 90% (P o0.05) at high concentration of PEG/LA-CS@Fe3O4 NPs after 7 days incubation. These results illustrate that PEG/LACS@Fe3O4 NPs have excellent cytocompatibility and are possible for application in vivo.

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vitro after standing for one month (Fig. 8a for T2WI and Fig. 8b for T1W1). It can be seen that the signal is uniformity and there is no magnetization artifact. Also, the relative signal strength at different levels of the tubes is shown in Table 1. Likewise, there is no significant difference at the different levels for both T2W1 and T1W1 sequence. These results further confirm that the PEG/LACS@Fe3O4 NPs have good dispersion stability and are beneficial for in vivo application. For in vivo MRI, the PDWI MRI of the livers of SD rates before and 30 min after intravenous administration of PEG/LA-CS@Fe3O4 NPs is shown in Fig. 9. The parenchymal signal strength of the rate livers is uniform and there is no obvious difference with the other organs before intravenous administration of PEG/LA-CS@Fe3O4 NPs (Fig. 9a). However, the parenchymal signal strength of rate liver significantly decreases after PEG/LA-CS@Fe3O4 NPs administration (Fig. 9b), strongly proving that PEG/LA-CS@Fe3O4 NPs can be used in liver MRI as negative contrast agent and also indicating that PEG/LA-CS@Fe3O4 NPs has liver targeting function. 3.4. Metabolism and iron toxicity of PEG/LA-CS@Fe3O4 NPs As a contrast agent, PEG/LA-CS@Fe3O4 NPs should be metabolized by the body, either through absorption or excretion and also should be non-toxic. Then the metabolism and iron toxicity of PEG/LA-CS@Fe3O4 NPs for the rate were tested through intravenous administration of serious overdose of PEG/LA-CS@Fe3O4 NPs. Fig. 10 shows the liver MRI of the rate after intravenous administration of serious overdose of PEG/LA-CS@Fe3O4 NPs at different time. As shown in Fig. 10, the parenchymal signal strength

3.3. . In vitro and in vivo MRI of PEG/LA-CS@Fe3O4 NPs Before in vivo MRI study, the in vitro MRI was first investigated to determine the performance, especially the dispersion stability of the contrast agent. Fig. 8 is the MRI of PEG/LA-CS@Fe3O4 NPs in

Fig. 11. Prussian blue histological staining of adjacent liver tissue slices after overdose injection of PEG/LA-CS@Fe3O4 NPs 24 h (a) and 7 days (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Center of Yangzhou University, Jiangsu Province Postdoctoral Science Foundation and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

References

Fig. 12. Pathological examination of the liver tissue after overdose injection of PEG/ LA-CS@Fe3O4 NPs 7 days.

of the rate livers significantly decreases compared with that before administration of PEG/LA-CS@Fe3O4 NPs. However, the decrease for 7 days is lower than that of 24 h, indicating that PEG/LACS@Fe3O4 NPs can be metabolized by the rate liver and also suggesting that no damage on the liver function occurred within the tested time. Prussian blue staining was also used to confirm the uptake and metabolism of PEG/LA-CS@Fe3O4 NPs by liver tissues (Fig. 11). Blue color shows the aggregated PEG/LA-CS@Fe3O4 NPs in livers. Obviously, there were still PEG/LA-CS@Fe3O4 NPs after 7 days injection, suggesting the circulation time is relatively long and is consistent with that PEG can prolong the circulation time in vivo [15]. However, the amount of PEG/LA-CS@Fe3O4 NPs in the liver after 7 days is less than that after 24 h, demonstrating that PEG/LACS@Fe3O4 NPs can be uptaken and metabolized by liver and indirectly indicate it is not toxic to the liver. These results are consistent with that from MRI studies above. To further confirm the non-toxicity of PEG/LA-CS@Fe3O4 NPs to liver, the histopathological analysis of the liver tissue 7 days after intravenous administration of serious overdose of PEG/LACS@Fe3O4 NPs was carried out (Fig. 12). It can be seen that the liver is normal and no lesions performance occurs, directly suggesting the safety of the PEG/LA-CS@Fe3O4 NPs to liver.

4. Conclusions Biofunctionalized PEG/LA-CS@Fe3O4 NPs were prepared and characterized. To value the application of the developed PEG/LACS@Fe3O4 NPs, its dispersion stability, magnetic properties, biocompatibility, MRI, metabolism and toxicity were investigated. As a result, the coating of Fe3O4 NPs with PEG/LA-CS can dramatically improve its dispersibility in physiological medium and the newly developed PEG/LA-CS/Fe3O4 NPs was confirmed to be an effect negative contrast agent for liver MRI in terms of the good dispersibility, biocompatibility, MRI stability and non-toxicity.

Acknowledgements This research was supported by National Natural Science Foundation of China (No. 21201149, No. 51073133), the Natural Science Foundation of Jiangsu Province (BK2012259), the Testing

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