N-hexanoyl chitosan stabilized magnetic nanoparticles: Implication for cellular labeling and magnetic resonance imaging
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This project involved the synthesis of N-hexanoyl chitosan or simply modified chitosan (MC) stabilized iron oxide nanoparticles (MC-IOPs) and the biological evaluation of MC-IOPs. IOPs containing MC were prepared using conventional methods, and the extent of cell uptake was evaluated using mouse macrophages cell line (RAW cells). MC-IOPs were found to rapidly associate with the RAW cells, and saturation was typically reached within the 24 h of incubation at 37°C. Nearly 8.53 ± 0.31 pg iron/cell were bound or internalized at saturation. From these results, we conclude that MC-IOPs effectively deliver into RAW cells in vitro and we also hope MC-IOPs can be used for MRI enhancing agents in biomedical fields.
KeywordsChitosan High Resolution Transmission Electron Microscopy Dynamic Light Scattering High Resolution Transmission Electron Microscopy Iron Oxide Nanoparticles
Magnetic particles ranging from the nanometer to micrometer scale are being used in an increasing number of medical applications. The important properties of magnetic particles for medical applications are nontoxicity, biocompatibility, injectability, and high level accumulation in the target tissue or organ; the most important property among those mentioned above is nontoxicity. Magnetic nanoparticles offer attractive and versatile applications in the field of biotechnology, such as DNA and RNA separation, cell separation, drug delivery system (DDS), magnetic resonance imaging (MRI), and hyperthermia [1, 2, 3, 4, 5, 6]. For these applications, magnetic iron oxides such as Fe3O4 or gamma-Fe2O3 are employed as a magnetic phase because they are stable and harmless to the living bodies. To make them bind to a biological entity, their surfaces are usually modified with an appropriate compound such as polyethyleneglycol (PEG) or streptavidin. Polymers like poly-L-lysine (PLL), poly ethylene imide (PEI) and dextran, and recently chitosan  has been used as a stabilizer (coating agent) for iron oxide nanoparticles so as to improve the nanoparticle's biocompatibility and injectability. However, high-level accumulation in the target tissue or organ and cytotoxicity; the most important property of the nanoparticles is remains to be intact.
More or less to improve limitations stated above, several derivatives of chitosan have been proposed based on reactions with the free amino groups. Our research group already investigated the hydrophobic modification of natural chitosan by using three different acyl chlorides (hexanoyl, octanoyl and myristoyl chloride) so as to improve its aqueous solubility and subsequently used them for stabilization of metalic nanoparticles [7, 8, 9]. In this paper, we have selected the hexanoyl chloride modified chitosan stabilized iron oxide nanoparticles (N ac-6-IOPs or simply MC-IOPs) as a material of interest and demonstrated its biomedical application like cellular labeling, and MRI using mouse macrophages cell line (RAW cells).
Results and discussion
Synthesis and characterization of MC-IOPs
Physiochemical properties of IOPs and MC-IOPs
Biocompatibility and cellular labeling of MC-IOPs
Magnetic resonance (MR) study of MC-IOPs
MC-IOPs synthesized by simple precipitation method showed highly crystalline, superparamagnetic behavior. It also displayed high stability, nontoxicity, enhancement of MR images and the potential endocytose the macrophage cell line. From above preliminary results, we conclude that MC-IOPs could be a better candidate for MR contrast medium.
Iron (III) chloride hexahydrate (FeCl3 6H2O) pure granulated, 99%, iron (II) chloride tetrahydrate (FeCl2 4H2O) 99+%, and ammonium hydroxide (14.8 M) were purchased from Fisher Scientific (Pittsburgh, PA). Deionized water purged with nitrogen gas was used in all the steps involved in the synthesis and formulation of iron oxide nanoparticles. Chitosan-100 [viscosity average molecular weight, Mv = 1.3 × 106, degree of deacetylation (fraction of free amino group) 78%] was purchased from Wako Pure Chemical Industries, Ltd., Japan.
Synthesis of iron oxide nanoparticles (IOPs)
Aqueous solutions of 0.1 M Fe(III) (30 mL) and 0.1 M Fe(II) (15 mL) were mixed, and 3 mL of 5 M ammonia solution was added dropwise over 1 min while stirring on a magnetic stir plate. The stirring continued for 20 min under a nitrogen-gas atmosphere. The particles obtained were washed 3 times using ultracentrifugation (25000 × g for 20 min at 4°C) with nitrogen purged water. The iron oxide nanoparticle yield, determined by weighing of the lyophilized sample of the preparation, was 304 mg.
Modification of chitosan (MC)
The modification process of chitosan was taken from a previously described report [7, 8, 9]. Briefly, a mixture of chitosan-100 (0.83 g) and 1.0% aqueous acetic acid (100 ml) was stirred for 24 h to ensure total solubility. The pH was adjusted to 7.0 by slow addition of 0.1 M of NaOH with strong agitation, yielding gel slurry. After addition of 0.02 M of fatty acyl chloride (hexanoyl chloride, FW = 134.61, d = 0.978 g/ml), the resultant solution was diluted 11 times with de-ionized water. After 6 h of continuous stirring, the solution was neutralized (pH 6.8–7.0) by 0.1 M of NaOH and precipitated with acetone. The precipitate, collected by filtration, was washed at 50–60°C with an excess of methanol and decanted. The washing was repeated 4 times to eliminate free fatty acids. Finally, the products were dried under vacuum for 3 days at room temperature. The chemical structure of native and modified chitosan is shown in Figure 1.
Stabilization of iron oxide nanoparticles (MC-IOPs)
Polymer (5.0 ml of 0.33% of N-hexanoyl chitosan solutions or MC) was added to the dispersion of the nanoparticles (100 mg) (the dispersion was cooled to room temperature but not lyophilized) and stirred overnight in a closed container to minimize exposure to atmospheric oxygen to prevent oxidation of the IOPs. These particles were washed with nitrogen purged water to remove soluble salts and excess polymer. Particles were separated by ultracentrifugation at 30000 rpm (Optima LE-80K, Beckman, Palo Alta, CA) using a fixed angle rotor (50.2 Ti) for 30 min at 10°C. The supernatant was discarded, and the sediment was redispersed in 15 mL of triply distilled water by sonication in a water-bath sonicator (FS- 30, Fisher Scientific) for 10 min. The suspension was centrifuged as above, and the sediment was washed three times with triply distilled water. Nanoparticles were resuspended in triply distilled water by sonication as above for 20 min and centrifuged at 1000 rpm for 20 min at 7–11°C to remove any large aggregates. The supernatant containing MC-IOPs was collected and re-diluted in phosphate buffer at pH 7.4.
Structural characterization of MC-IOPs
FT-IR spectra were recorded at RT using a Perkin-Elmer spectrometer, model 2000. The FT-IR spectrometer was linked to a personal computer loaded with the IRDM (IR Data Manager) program to process the recorded spectra. The specimens were pressed into small discs using a spectroscopically pure KBr matrix. FT-IR measurements were checked by the X-ray diffraction of isolated precipitates. XRD (APD-10, Philips, Netherlands) was performed to identify the structure of the MC-IOPs using Cu K alpha radiation (λ = 1.54056 Å) between 20° and 90° (2θ) at 27°C.
Particle size, morphology and ξ-potential analysis of MC-IOPs
The size and morphology of IOPs and MC-IOPs were observed by TEM (JEM-1230, JEOL, Japan) and HRTEM (QUANTA 200F, FEI, USA). The sample for TEM analysis was obtained by placing a drop of IOPs and MC-IOPs suspension diluted by distilled water onto a copper grid without any staining, and drying it in air at room temperature. The average hydrodynamic diameter and the ξ-potential of IOPs and MC-IOPs were determined by DLS and ELS (Zetasizer ZEN 3600, Malvern, UK), respectively. All DLS measurements were done with an angle detection of 90° at 25°C after diluting the dispersion to an appropriate volume with water. The results were the mean values of two experiments using the same sample.
Magnetic property of MC-IOPs
Magnetic measurement was done using a SQUID magnetometer (MPMSXL-7, Quantum Design, USA). Magnetization curves were recorded for a suspension and solid sample of MC-IOPs at 27°C with an applied magnetic field up to 10,000 Oe.
Evaluation of cytotoxicity
Cellular uptake of MC-IOPs
To test cell up take study, RAW cells were prepared and incubated at a concentration of 1 × 106 cells/ml with 5, 10 and 20 μl MC-IOPs (11.2 mg/ml stock) for 2 h, then incubated with fresh medium overnight. The cells were harvested and measured by flow cytometry using SSC signal. Similarly harvested RAW cells were further used for Prussain blue staining using K4 [Fe(CN)6] reagents. Iron determination was performed by colorimetric determination method.
Magnetic resonance (MR) study of MC-IOPs
For MR study, MC-IONPs were incubated with RAW cells at different concentration for 24 h. The cells were harvested and washed three times and centrifuged at the cell number 1 × 103. The cell plates were scanned using 1.5T MR system. Under T2 weighted MR images of MC-IONPs were obtained with 1.5T MR system (Medius Co. Korea, Model Magnum1.5T) by using a spin echo technique. The differences between MR images of cells with and without MC-IONPs incubation were compared.
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (the Center for Healthcare Technology Development, Chonbuk National University, Jeonju 561-756, Republic of Korea). We thank R. Lamichane of WSU for careful manuscript correction.
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