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J. Phys. Chem. C 2008, 112, 5432-5438
Preparation of Well-Dispersed Superparamagnetic Iron Oxide Nanoparticles in Aqueous Solution with Biocompatible N-Succinyl-O-carboxymethylchitosan Aiping Zhu,*,† Lanhua Yuan,† and Sheng Dai‡ College of Chemistry and Chemical Engineering, Yangzhou UniVersity, Yangzhou 225002, P. R. China, and National Institute for Nanotechnology, National Research Council Canada, Edmonton, Alberta T6G 2M9, Canada ReceiVed: NoVember 30, 2007; In Final Form: December 12, 2007
N-succinyl-O-carboxymethylchitosan (NSOCMCS), an amphiphilic polyelectrolyte with the property of biocompatibility and functional carboxyl groups, was used as a stabilizer to prepare a well-dispersed suspension of superparamagnetic Fe3O4 nanoparticles, which were composed of a magnetite Fe3O4 core and a NSOCMCS shell. The carboxyl groups of NSOCMCS can coordinate with Fe3O4, which makes NSOCMCS chemically adsorb onto the surface of Fe3O4 nanoparticles. The stabilizing mechanisms were proven to be both the steric hindrance and electrostatic repulsion arisen from the NSOCMCS. Transmission electron microscopy showed that the resulting Fe3O4 particles were in spherical morphology with the diameter ranging from 12 to 18 nm. Magnetic property measurements indicated that NSOCMCS/Fe3O4 nanoparticles preserved superparamagnetic behavior. In vitro biocompatibility studies showed that the NSOCMCS/Fe3O4 nanoparticles had good cytocompatibility.
1. Introduction Magnetic support-based separation has been widely applied in various fields of biotechnology and biomedicine, including cell separation,1 enzyme immobilization,2,3 drug delivery,4 and protein purification.5,6 In order to increase the delivery efficiency of magnetic particles and to improve their vascular residence time, the particle surface needs to be modified to minimize or delay the nanoparticle uptake by the mononuclear phagocyte system (MPS or reticuloendothelial system).7,8 The ideal magnetic supports for biotechnology applications should fulfill some necessary properties, such as superparamagnetism, strong magnetic responsiveness, high stability, narrow size distribution, and biocompatibility. The nanoparticles investigated for drug delivery, medical diagnostics, and imaging purposes always require a protective organic layer to prevent coagulation.9,10 Moreover, the magnetic particles should have a relatively high density of functional groups for coupling ligands or binding biomolecules. Surface functional groups could be introduced onto magnetic polymer hybrid particles by two methods: copolymerization and chemical modification of a preformed polymer. The copolymerization method cannot give nanoparticles with a high density of surface functional group since a large amount of functional groups will be buried inside the polymer.11 Chemical modification has been reported to be an efficient way to obtain sufficient functional groups on the surface of magnetic particles.12 One of the challenges facing researchers is to be able to transport and deliver well-dispersed magnetic nanoparticles with desired composition, structure, and uniformity. The principal cause of aggregation is the van der Waals attraction forces between the particles. To counteract these attractive interactions * Corresponding author. Tel: 86-514-7975568. Fax: 86-514-7975524. E-mail:
[email protected]. † Yangzhou University. ‡ National Institute for Nanotechnology.
and promote stability, equally repulsive forces are required.13 They could be achieved either by electrostatic repulsion between the particles or by coating the particles with organic layer molecules (adlayer) that act as dispersants.14-16 In a biomedical application, the magnetic nanoparticles could be coated with natural macromolecules (e.g., proteins and polysaccharides) and synthetic charged (e.g., polyelectrolytes) and nonionic polymers (e.g., polyvinyl alcohol).17-19 Among these adlayers, the natural macromolecules showed their prominent advantage due to biocompatibility and biodegradability. Chitosan is a polysaccharide formed primarily of repeating units of β-(1-4) 2-amino-D-glucose (glucosamine). Being a nontoxic, biocompatible, and biodegradable natural polymer, chitosan has been investigated for its pharmaceutical and biomedical applications.20-24 Moreover, the active hydroxyl and amino groups on the chitosan molecules favor the development of different chemical-derived modifications with tailored biophysical properties, e.g., amphiphilic properties,25 crystallinity,26 biomembrane fusion property,27 and cytocompatibilty.28 Chitosan derivatives have been shown to possess physicochemical and biological properties different from those of chitosan. For example, chitosan has high nonspecific protein adsorption resulting in a promotion of blood clotting, while O-carboxymethylchitosan has low nonspecific protein adsorption and proved to show good compatibility with blood.29 Therefore, it is predicted that a chitosan derivative with designed structure and biological properties will provide the possibility of being used as a novel dispersant to stabilize superparamagnetic iron oxide nanoparticles and endow its ideal biophysical properties to the iron oxide nanoparticles in biomedical applications. Though many papers deal with biocompatible superparamagnetic iron oxide nanoparticles due to their popular applications in drug delivery research, cellular-level intervention in disease conditions is far from developed. This kind of intervention requires the delivery platform to remain in systemic circulation
10.1021/jp711319a CCC: $40.75 © 2008 American Chemical Society Published on Web 03/18/2008
Superparamagnetic Iron Oxide Nanoparticles SCHEME 1: Synthesis Scheme of NSOCMCS
for a long period of time and also requires transportation of the devices from the blood stream into target tissues. A nanoparticle suspension stabilized by a biopolymer with the desired biophysical properties can provide the possibility of solving this problem. N-succinyl-O-carboxymethylchitosan (NSOCMCS) has been recently synthesized, and its physicochemical properties, especially its amphiphilic polyelectrolyte property, have been investigated.30 The synthesis scheme is shown in Scheme 1. The functional carboxyl acid groups are supposed to exhibit coordination interaction with Fe3O4, and the amphiphilic and polyelectrolytic properties are supposed to provide not only the electrostatic stabilization but also the steric hindrance to stabilize the suspension of iron oxide nanoparticles. The aim of this study is to demonstrate that NSOCMCS is an excellent dispersant to prepare well-dispersed superparamagnetic iron oxide nanoparticles simply in aqueous solution. The adsorption mechanism of NSOCMCS onto Fe3O4 nanoparticles and the stabilizing mechanism of Fe3O4 nanoparticles by NSOCMCS are discussed extensively. Experimental results demonstrate that NSOCMCS/ Fe3O4 nanoparticles possess good cytocompatibility, indicating their potential applications in biomedical fields. 2. Experimental Section 2.1. Chemicals and Materials. Ferric chloride hexahydrate (FeCl3‚6H2O, >99%), ferrous chloride tetrahydrate (FeCl2‚ 4H2O, >99%), and ammonium hydroxide (29.4 wt %) were obtained from Shanghai Chemical Reagent (China). Chitosan was obtained from Lianyungang Biologicals, Inc. (China). Its viscosity-averaged molecular weight was 5.2 × 105 g/mol, and the degree of deacetylation was 90%. O-carboxymethylchitosan, containing about 100 carboxyl groups and 75 amino groups per 100 anhydroglucosamine units of chitosan, was prepared by methods reported previously.25 Fresh Milli-Q water was used as the solvent. All the other chemicals were of reagent grade and used without further purification. 2.2. Preparation of N-Succinyl-O-carboxymethylchitosan (NSOCMCS). The synthesis of NSOCMCS was done according to our recent report.30 Briefly, 1 g of OCMCS was dissolved into 200 mL of distilled water and then transferred to a flask. Succinic anhydride (0.2 g) was dissolved in 20 mL of acetone and added to the flask dropwise for 30 min at room temperature, and then the reaction was allowed to react for 4 h at 40 °C. The reaction mixture was cooled to room temperature. The mixture was precipitated in an excess of ethanol, filtered to remove the solvent, and then washed with 70, 80, and 100% ethanol. Finally, the product was dried at 40 °C under vacuum for 24 h. The obtained white powder NSOCMCS had a mass of 1.1 g. The NSOCMCS solutions were prepared with Millipore deionized water.
J. Phys. Chem. C, Vol. 112, No. 14, 2008 5433 2.3. Preparation of Magnetic Colloid. The magnetic Fe3O4 nanoparticles were prepared without the addition of any stabilizer according to the following procedure. Typically, 5 mL of iron solution containing 0.1 M Fe2+ and 0.2 M Fe3+ was added slowly to 50 mL of NH4OH (29.4 wt %) solution under vigorous magnetic stirring and protection with N2 for 30 min at room temperature. The suspension color turned black immediately. The stirring was stopped, a strong magnet was used to settle the black precipitate, and the supernatant was then decanted. Double deionized and deoxygenated water was added to wash the powder. The solution was centrifuged for 2 min at a speed of 1800 rpm, and the supernatant was decanted. Centrifugation-redispersion cycles were carried out several times to remove excess ammonia from the remaining solution. Finally, a black precipitate (magnetite) was obtained by freezedrying. 2.4. NSOCMCS Surface-Modified Fe3O4 Nanoparticles. For the preparation of magnetic Fe3O4 nanoparticles stabilized by NSOCMCS, 10 mg of as-prepared Fe3O4 black precipitate was first dispersed into 10 mL of NSOCMCS aqueous solutions with known concentration (e.g., 0.2 mg/mL), then stirred for 12 h at room temperature to ensure that the Fe3O4 nanoparticles were evenly coated. The black dispersions were purified by centrifugation and redispersion cycles for several times to remove free NSOCMCS molecules. The colloidal solution of NSOCMCS/ Fe3O4 magnetic nanoparticles were centrifuged for 10 min at a speed of 25 000 rpm. Finally, NSOCMCS/Fe3O4 nanoparticles were obtained by freeze-drying. The suitable concentration of NSOCMCS for stabilization of Fe3O4 was found to be in the range of 0.1-0.5 mg/mL. When the concentration was lower than 0.1 mg/mL, the number of NSOCMCS molecules was not enough to stabilize the iron oxide nanoparticles, while too high of a concentration of NSOCMCS will produce its aggregates and thus decrease the coating efficacy on the surface of Fe3O4 nanoparticles. As a result, 0.2 mg/mL of NSOCMCS was used in the preparation procedure. 2.5. Characterization. The crystal structures of the samples were examined by X-ray diffraction (XRD) with a XD-3A powder diffractometer, using a monochromatized X-ray beam with nickel-filtered Cu KR radiation in the range of 5-40° (2θ) at 40 kV and 30 mA. The size and morphology of the magnetic nanoparticles were monitored by a TE CHAI-12 (Philips) transmission electron microscopy (TEM) instrument. The particle size distribution was investigated using a commercial LLS system (ALV/SP-125, Germany), which was equipped with a solid-state laser (Coherent DPSS) with an output power of 400 mW at λ0 532 nm and an ALV-5000 multi-τ digital time correlator. The magnetization and hysteresis loop were measured at room temperature with a Lake Shore model 7300 VSM. The ζ potential of the magnetic nanoparticles was measured using a ζ-potential analyzer (Zetaplus, Brookhaven Instruments, USA). 2.6. In Vitro Cell Viability/Cytotoxicity Studies. Fibroblast cells (3T3) (NIH/3T3) (ACTT CRL-1658) were cultured in Dulbecco’s modified Eagle’s medium media containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C with a 5% CO2 environment. The media was changed every third day. The cells were plated at a density of 1 × 104 cells/well in a 96-well plate at 37 °C in a 5% CO2 atmosphere. After 12 h of culture, the media in the well was replaced with fresh media containing Fe3O4 nanoparticle concentrations of 0-300 µg/mL. The media was removed, and the cells were washed three times with phosphate-buffered saline (PBS) to remove the nanoparticles after 72 h. Then 20 µL of combined MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-
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Figure 1. TEM morphologies of (a) unmodified Fe3O4 nanoparticles, (b) NSOCMCS-modified Fe3O4 nanoparticles at pH 7.4, and (c) NSOCMCSmodified Fe3O4 at pH 9.0.
yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/PMS (phenazine methosulfate) solution (Promega) was added to each well of the 96-well assay plate containing 100 µL of cell culture media. After incubation of the plate for 2 h at 37 °C in a humidified, 5% CO2 atmosphere, the absorbance of each well at 490 nm was recorded by using a 96-well plate reader (Bio-Rad Labs). The spectrophotometer was calibrated to zero absorbance using culture media without cells. The relative cell viability related to control wells containing cell culture media without the nanoparticles was calculated by [A]test/[A]control, where [A]test is the absorbance of the test sample and [A]control is the absorbance of control sample. The experiment was repeated three times. The statistical analysis of experimental data utilized the student’s t test, and the results were presented as mean SD. Statistical significance was accepted at a level of p < 0.05. 3. Results and Discussion 3.1. Characterization of Fe3O4 Nanoparticles. 3.1.1. TEM Morphology. TEM micrographs of unmodified Fe3O4 nanoparticles and NSOCMCS/Fe3O4 nanoparticles obtained from 0.2 mg/mL NSOCMCS solution in different pH values (such as 7.4 and pH 9) are shown in Figure 1. The images illustrate that the magnetic Fe3O4 nanoparticles are spherical or ellipsoidal
with a mean particle size of 14 nm associated with a standard deviation of 3 nm (Figure 1a). For NSOCMCS/Fe3O4 nanoparticles (Figure 1b), the average particle size and standard deviation are 17 and 3 nm, respectively, prepared atpH 7.4. It also illustrates the core-shell structure of NSOCMCS/Fe3O4 nanoparticles with the magnetite inner core and NSOCMCS outer shell. The thickness of the NSOCMCS shell is about 2-3 nm. In the case of pH 9 (Figure 1c), the average particle size and standard deviation are 16 and 4 nm, respectively, and all nanoparticles show well-dispersed spherical images. However, the NSOCMCS shell could not be detected because the thickness may be lower than the detection limit of the TEM instrument. Such results indicate that pH affects the thickness of the adlayer outside Fe3O4 nanoparticles. The images of NSOCMCSmodified Fe3O4 nanoparticles clearly illustrate that these nanoparticles exhibit well-dispersed morphology and only a small number of nanoparticles are aggregated. 3.1.2. XRD. To confirm the presence of crystalline structure for unmodified Fe3O4 and NSOCMCS/Fe3O4 nanoparticles, XRD was used. The diffractograms are shown in Figure 2. There are six diffraction peaks: (220), (311), (400), (422), (511), and (440) in the unmodified Fe3O4 particles, which is the standard pattern for crystalline magnetite with spinel structure.31 At the
Superparamagnetic Iron Oxide Nanoparticles
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Figure 4. ζ Potential of Fe3O4 and NSOCMCS/Fe3O4 nanoparticles in solution.
Figure 2. X-ray powder diffraction patterns of Fe3O4 and NSOCMCS/ Fe3O4 nanoparticles.
Figure 5. Photographs of the suspensions obtained from pH levels of 9, 7.4, and 4 in 0.2 mg/mL of NSOCMCS solution and unmodified Fe3O4 nanoparticles from left to right.
Figure 3. Magnetization curves for Fe3O4 and NSOCMCS/Fe3O4 nanoparticles at room temperature.
same time, it is obvious that the diffraction pattern for NSOCMCS-modified Fe3O4 particles is similar to that of unmodified Fe3O4 nanoparticles. Such result suggests that the crystalline structure of Fe3O4 does not change with NSOCMCS stabilization. 3.1.3. VSM. The saturation magnetization (MS) values were normalized by assuming 100% magnetite for simplicity, using the iron mass as determined by atomic absorption spectroscopy.32 Figure 3 shows typical hysteresis curves at 300 K for the optimized nanoparticle formulation. The hysteresis loops have negligible coercivity at room temperature. The synthesized magetites indicate a superparamagnetic behavior as evident by zero coercivity and remanance on the magnetization loop. The saturation magnetization of unmodified Fe3O4 nanoparticles and NSOCMCS-modified Fe3O4 nanoparticles obtained from pH levels of 4, 7.4, and 9 of NSOCMCS solution was determined to be 59.0, 53.4, 52.7, and 59.5 emu/g, respectively. These results indicate that the adlayer of NSOCMCS molecules has little influence on the MS value of Fe3O4 nanoparticles. This is the advantage of this system, different from other methods to prepare polymer-modified magnetites, in which the saturation magnetization was found to be only 16.3 emu/g, caused by the low Fe3O4 content (24.3%) in the modified Fe3O4 nanoparticles.31 Moreover, the fabrication process is tedious.33,34
3.2. Physicochemical Properties of Suspension of Fe3O4 Nanoparticles. Associated with important biomedical applications including ferrofluids, magnetic resonance imaging, and magnetically guided drug delivery, one of the main challenges is to fabricate and deliver well-dispersed magnetic nanoparticles with the desired composition and structure. Since magnetic nanoparticles have the tendency to aggregate, the introduction of dispersant is required in order to counteract these attractive interactions and promote stability. And the stabilization mechanism was strongly dependent on the physicochemical properties of the dispersant. 3.2.1. Fe3O4 Nanoparticles Stabilized by NSOCMCS. Dynamic light scattering (DLS) measurements were carried out to investigate the particle size and its distribution of the resulting NSOCMCS-stabilized Fe3O4 nanoparticles. The hydrodynamic radius distribution of unmodified and NSOCMCS-stabilized Fe3O4 nanoparticles at a scattering angle of 90° is shown in Figure 6. The averaged apparent hydrodynamic radii and polydispersity indexes of NSOCMCS modified-Fe3O4 nanoparticles formed at pH levels of 4, 6.5, 7.4, and 9 were found to be 290, 68, 42, and 53 nm and 0.342, 0.267, 0.295, and 0.241, respectively, while the average radius of and polydispersity index for the unmodified Fe3O4 nanoparticles was 748 nm and 0.05, respectively, which suggests a very fast and strong aggregation. From DLS measurement, it can be concluded that NSOCMCS is an effective dispersant to stabilize Fe3O4 nanoparticles. The size of the NSOCMCS-modified Fe3O4 nanoparticles is strongly dependent on pH, and it seems that high pH (6.5-9.0) favors the formation of stable NSOCMCS-modified Fe3O4 nanoparticles.
5436 J. Phys. Chem. C, Vol. 112, No. 14, 2008
Figure 6. Hydrodynamic radius distributions of Fe3O4 and NSOCMCS/ Fe3O4 nanoparticles from DLS.
Due to the amphiphilic character of NSOCMCS, it aggregates in dilute solution and the averaged radius of the aggregates was found to be 48 nm (pH 7.4 and 0.01 M NaCl solution). When compared with the apparent radius of the NSOCMCS-modified Fe3O4 nanoparticles at the same experimental conditions (pH and salt concentration), it can be concluded that the NSOCMCS adsorbed on the surface of Fe3O4 nanoparticles should be in the form of a multilayer deposit rather than aggregates. However, at low pH (∼4), the NSOCMCS-modified Fe3O4 shows a larger radius of 290 nm, which indicates that the adsorbed NSOCMCS may be aggregated. In comparison with the TEM morphologies shown in Figure 1, it is easy to find that the hydrodynamic radius measured by DLS (Figure 6) is much larger. The large size of the hydrodynamic radius should be due to the solvation between water and NSOCMCS. In aqueous solution, there are electrostatic repulsive interactions among the dissociated carboxylic acid groups on the surface of Fe3O4 nanoparticles. Moreover, there are amide groups on the NSOCMCS-modified Fe3O4 nanoparticles, which lead to hydrogen bonding interactions and thus the solvation between water and NSOCMCS-modified Fe3O4 nanoparticles. The size measured by TEM is very small, which is caused by the shrinkage associated with the drying process. To further investigate the stability of NSOCMCS/Fe3O4 nanoparticles, all above suspensions were centrifuged at 10 000 rpm for 10 min and then their pictures were taken (Figure 5). From left to right, these pictures are NSOCMCS/Fe3O4 nanoparticles obtained from pH levels of 9.0, 7.4, and 4.0 and unmodified Fe3O4 nanoparticles in deionized water, respectively. The suspensions of NSOCMCS/Fe3O4 nanoparticles prepared from pH levels of 7.4 and 9.0 exhibit a transparent colloidal appearance, which demonstrates the extremely stable colloidal solution. The suspension of NSOCMCS/Fe3O4 nanoparticles obtained from pH 4 shows an opaque appearance, which suggests that a certain amount of larger particles have been formed and precipitated. However, the suspension of unmodified Fe3O4 nanoparticles exhibits two phases; the bottom phase is the precipitate of Fe3O4 nanoparticles, and the supernatant is water, which confirms that the superparamagnetic nanoparticles without a protective layer are easy to aggregate in solution. The stabilization tests further indicate that NSOCMCS is one type of effective stabilizer to protect Fe3O4 nanoparticles from aggregating in solution. Besides, NSOCMCS has also been proven to inhibit oxidation effectively even for long-time
Zhu et al. exposures at ambient conditions, and the NSOCMCS/Fe3O4 nanoparticles can retain their initial properties more than 6 months. 3.2.2. Adsorption of NSOCMCS on Fe3O4 Nanoparticles. The stabilization of Fe3O4 nanoparticles by NSOCMCS is due to the adsorption of NSOCMCS onto the surface of Fe3O4 nanoparticles. As seen from the NSOCMCS structure (Scheme 1), there are two carboxyl acid groups (-COOH) on each unit of NSOCMCS. As a result, NSOCMCS can adsorb on Fe3O4 nanoparticles with the oxygen atoms of the carboxylate group coordinated symmetrically with the iron atoms of Fe3O4. This adsorbed layer of NSOCMCS forms the outer shell, with magnetite as the inner core, and has been confirmed by TEM micrographs (Figure 1b), in which a NSOCMCS adlayer of 2-3 nm thickness encapsulates Fe3O4 nanoparticles. At a higher pH of 9, the thickness of the NSOCMCS adlayer becomes thinner compared with that at pH 7.4. It has been reported that PEG could be used to effectively stabilize Fe3O4 nanoparticles. However, the functional groups such as 1,2,3-trihydroxybenzene groups (pyrogallol)35 or the oligo(aspartic acid) carboxylate36 should be introduced to the PEG molecules first. These functional head groups are supposed to adsorb onto amphoteric oxide surfaces via ligand exchange reactions; however, the procedure are tedious. Poly(vinyl alcohol) (PVA) is also known to be a good stabilizer for Fe3O4 nanoparticles, promoted by the hydrogen bonding between the polymer and nanoparticles.37 In the present system, NSOCMCS is an amphiphilic polyelectrolyte composed of two carboxylic acid groups in each repeating unit. Therefore, the density of functional groups of NSOCMCS is much higher than that of end-group-functionalized polyethylene glycol (PEG) and sharply increases the adsorption capability of NSOCMCS onto Fe3O4 nanoparticles accordingly. As a result, a thin NSOCMCS adlayer is enough to stabilize the Fe3O4 nanoparticles. The NSOCMCS-stabilizing behavior of Fe3O4 nanoparticles is strongly dependent on pH, which is attributed to the polyelectrolyte property of NSOCMCS. With increasing pH, more and more carboxylic acid groups are neutralized and increase the negative charges and the hydrophilicity of NSOCMCS, since the un-neutralized carboxylic acid groups are expected to produce coordination interaction with Fe3O4 more efficiently than that of neutralized carboxylic acid groups. As a result, the thickness of the chemisorbed NSOCMCS shell in a moderate pH of 7.4 is thicker than that in a high pH of 9 as verified by TEM micrographs. In the solution of pH 4, few carboxyl groups disassociate, and NSOCMCS shows hydrophobic properties. In such a situation, NSOCMCS molecules adsorbed on Fe3O4 nanoparticles are not in multilayers but aggregates, which results in a large radius of the NSOCMCS/Fe3O4 nanoparticles. 3.2.3. Stabilizing Mechanism. Colloidal processing of magnetic nanoparticles has been extensively studied with an emphasis on the characteristics of superparamagnetic Fe3O4 nanoparticles dispersion as a ferrofluid. Suspensions of superparamagnetic Fe3O4 nanoparticles have van der Waals forces and magnetic dipole-dipole interactions generated from residual magnetic moments, which tends to agglomerate the particles. Therefore, to form a stable dispersion, repulsive forces are required to keep each particle discrete and prevent it from amassing as larger and faster setting agglomerates. Steric hindrance plays an important role in stabilizing suspensions, which is accomplished by the protective shields on the oxide surface produced by molecules or polymers.38 “Steric” Stabilization. The uncharged PEG and PVA are good dispersants to stabilize Fe3O4 nanoparticles with the driving force
Superparamagnetic Iron Oxide Nanoparticles of steric stabilization.39,40 When the polymer layer (e.g., PEG) is thick enough, the van der Waals attraction is negligible, leading to extra repulsive force. The overlap of polymer layers reduces the volume available and increases the energy barrier, producing a strong repulsive force among particles.41 In the NSOCMCS-stabilizing system, the adsorbed NSOCMCS on Fe3O4 nanoparticles extends the aqueous medium outward with its hydrophilic domains (especially the dissociated carboxylic acid groups). The suspension stability of all NSOCMCS/Fe3O4 nanoparticles is improved significantly as compared with that of unmodified Fe3O4 nanoparticles, confirming the steric stabilizing mechanism of the NSOCMCS adlayer. Electrostatic Stabilization. The suspension stability of NSOCMCS/ Fe3O4 nanoparticles is dependent on pH, which is caused by the polyelectrolyte property of NSOCMCS. ζ Potential is considered a key parameter to investigate the surface charge of Fe3O4 nanoparticles. The ζ potential for the unmodified Fe3O4 nanoparticles in water was measured to be -13.4 mV, which is not sufficient to prevent particle agglomeration and results in an unstable suspension (Figure 5). The ζ potentials of NSOCMCS/Fe3O4 suspensions with different pH levels are shown in Figure 4. The ζ potentials of NSOCMCS/Fe3O4 nanoparticles prepared from pH levels 4, 6.5, 7.4, and 9 were -16.5, -28.9, -34.5, and -45 mV, respectively, indicating that the negative charges on the NSOCMCS/Fe3O4 nanoparticles increase with an increase in pH, because more carboxyl acid groups are neutralized at high pH. The dissociated carboxyl groups provide enough charge to stabilize Fe3O4 nanoparticles. This is the reason that with higher pH levels (6.5, 7.4, and 9) well-dispersed suspensions of Fe3O4 nanoparticles can be obtained, but not in a pH level of 4. With a pH level of 4, the ζ potential of NSOCMCS/Fe3O4 nanoparticles is -16.5 mV, as the negative charge is little higher than that of unmodified Fe3O4 nanoparticles. Therefore, the better dispersion of NSOCMCS/Fe3O4 nanoparticles with a pH level of 4 than that in unmodified Fe3O4 nanoparticles is not caused by the electrostatic stabilizing mechanism but by the steric stabilization mechanism. Promoted by both electrostatic stabilization and steric stabilization mechanisms, the adlayer of NSOCMCS in high pH levels is thus able to stabilize the suspension of Fe3O4 nanoparticles. The electrostatic repulsive force results from the electric double layer around particles, and thus, it is dependent on both pH and ionic strength of the suspension. The increase in ionic strength leads to an enhanced screening of particle surface charge and subsequently reduces the thickness of the Debye layer around particles. The addition of salt to the dispersion should initiate aggregation by suppressing the double layer. According to Derjaguin, Landau, Verwey, and Overbeek theory, below the critical coagulation concentration (CCC), the thickness of the electrical double-layer repulsion decreases with the increase of salt concentration. The double layer is entirely suppressed above the CCC, and the aggregation rate is then independent of salt concentration (rapid aggregation).42 In present system, the suspension stability of NSOCMCS/Fe3O4 nanoparticles is found to be not sensitive to the salt concentration. The above interesting result might be related to the strong adsorption of NSOCMCS on Fe3O4 nanoparticles leading to steric stabilization in addition to electrostatic stabilization. The softness of NSOCMCS in solution increases with ionic strength, which increase the adsorption on Fe3O4 nanoparticles to form the NSOCMCS adlayer. Therefore, for the NSOCMCS-stabilizing system, suitable ionic strength (0.1 M) is required to prepare a well-dispersed suspension of Fe3O4 nanoparticles.
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Figure 7. MTS assay viability of 3T3 cells incubated with NSOCMCS/ Fe3O4 nanoparticles for 72 h.
OCMCS can also stabilize the suspension of Fe3O4 nanoparticles driven mainly by electrostatic repulsion, as reported by our recent paper.43 The extra amide groups and hydrophobic moieties (-CH2CH2-) are introduced on the chains of NSOCMCS with the N-acylation of OCMCS with succinic anhydride. As a result, NSOCMCS is more easily adsorbed to the surface of Fe3O4 nanoparticles by the formation of chelation complexation in comparison with OCMCS. Therefore, NSOCMCS is more effective in stablization of the suspension of Fe3O4 nanoparticles than OCMCS. 3.3. Cytotoxicity. According to the experiment testing in vitro cell toxicity (data not shown), the cell compatibility of NSOCMCS is a little better than that of OCMCS. It is well-known that OCMCS is a typeof nontoxic biomaterial;28 therefore, NSOCMCS is another type of nontoxic chitosan derivative. Figure 7 shows the effect of Fe3O4 nanoparticles on cell viability. It can be seen that NSOCMCS/Fe3O4 nanoparticles have almost no effect on the cell viability compared with the that of the control. MTS assay data for the unmodified Fe3O4 nanoparticles was not obtained because they precipitated and adsorbed on cells. The adsorbed Fe3O4 nanoparticles could not be removed by washing and interfered with the MTS assay. NSOCMCS has been proved to be a good dispersant to prepare a well-dispersed suspension of Fe3O4 nanoparticles. Moreover, the functional carboxyl groups can be incorporated to drugs to provide time-specific and site-specific drug delivery through NSOCMCS/Fe3O4 nanoparticles. These versatile biological properties of NSOCMCS/Fe3O4 nanoparticles could be further explored for different biomedical purposes, for instance, the lubrication of surfaces in aqueous media, and the protection and targeting of particles for drug delivery, biological separation, and medical imaging. 4. Conclusions NSOCMCS, a biocompatible chitosan derivative, has been proved to be an excellent dispersant to prepare a well-dispersed suspension of superparamagnetic Fe3O4 nanoparticles due to its amphiphilic polyelectrolyte property. NSOCMCS can be easily chemisorbed onto Fe3O4 nanoparticles by the formation of chelation complexation. The adsorbed NSOCMCS stabilizes the Fe3O4 nanoparticles, a process which is driven by both steric and electrostatic stabilization mechanisms. Moreover, the NSOCMCS/ Fe3O4 nanoparticles have been proven to have good cytocompatibility, which is important for further biomedical
5438 J. Phys. Chem. C, Vol. 112, No. 14, 2008 applications. NSOCMCS, with functional carboxyl groups, can be incorporated in drugs to provide time-specific and sitespecific drug delivery through the modification of the NSOCMCS shell on Fe3O4 nanoparticles with stimuli-responsive functional groups or specific tissue-targeting ligands. Acknowledgment. This research was supported by a Natural & Scientific grant of Jiangsu Province, China, Project BK2006072 and No. 05KJB430149. References and Notes (1) Shen, L.; Laibinis, P. E.; Hatton, T. A. Langmuir 1999, 15, 447453. (2) Sun, S.; Zeng, H. J. Am. Chem. Soc. 2002, 124, 8204-8205. (3) Kang, Y. S.; Risbud, S.; Rabolt, J. F.; Stroeve, P. Chem. Mater. 1996, 8, 2209-2211. (4) Mann, S.; Hannington, J. P. J. Colloid Interface Sci. 1988, 122, 326-335. (5) Kim, D. K.; Zhang, Y.; Voit, W.; Rao, K. V.; Muhanned, M. J. Magn. Magn. Mater. 2001, 225, 30-36. (6) Zhang, Y.; Kohler, N.; Zhang, M. Biomaterials 2002, 23, 15531561. (7) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. ReV. 2001, 53, 283-318. (8) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. J. Mater. Chem. 2004, 14, 2161-2175. (9) Kohler, N.; Sun, C.; Wang, J.; Zhang, M. Q. Langmuir 2005, 21, 8858-8864. (10) Bertorelle, F.; Wilhelm, C.; Roger, J.; Gazeau, F.; Menager, C.; Cabuil, V. Langmuir 2006, 22, 5385-5391. (11) Khng, H. P.; Cunliffe, D.; Davies, S.; Turner, N. A.; Vulfson, E. N. Biotechnol. Bioeng. 1998, 60, 419-424. (12) Xie, X.; Zhang, X.; Zhang, H.; Chen, D. P.; Fei, W. Y. J. Magn. Magn. Mater. 2004, 277, 16-23. (13) Bonnemann, H.; Richards, R. M. Eur. J. Inorg. Chem. 2001, 10, 2455-2480. (14) Colfen H. Macromol. Rapid Commun. 2001, 22, 219-252. (15) Pastoriza-Santos, I.; Liz-Marzan, L. M. Langmuir 2002, 18, 28882894. (16) Sehgal, A.; Lalatonne, Y.; Berrett, J. F.; Morvan, M. Langmuir 2005, 21, 9359-9364. (17) Guo, L.; Pei, G. L.; Wang, T. J.; Wang, Z. W.; Jin, Y. Colloids Surf., A 2007, 293, 58-62. (18) Thode, K.; Luck, M.; Schroder, W.; Semmler, W.; Blunk, T.; Muller, R. H.; Kresse, M. J. Drug Targeting 1997, 5, 35-43.
Zhu et al. (19) Abu-Much, R.; Meridor, U.; Frydman, A.; Gedanken, A. J. Phys. Chem. B 2006, 110, 8194-8203. (20) Kofuji, K.; Ito, T.; Murata, Y.; Kawashima, S. Biol. Pharm. Bull. 2001, 24, 205-208. (21) Thanou, M.; Verhoef, J. C.; Junginger, H. E. AdV. Drug DeliVery ReV. 2001, 52, 117-126. (22) Roy, K.; Mao, H. Q.; Huang, S. K.; Leong, K. W. Nat. Med. 1999, 5, 387-391. (23) Mao, H. Q.; Roy, K.; Truong-Le, V. L.; Lin, K. Y.; Wang, Y.; August, J. T.; Leong, K. W. J. Controlled Release 2001, 70, 399-421. (24) Artursson, P.; Lindmark, T.; Davis, S. S.; Illum, L. Pharm. Res. 1994, 11, 1358-1361. (25) Zhu, A. P.; Chan, M. B.; Dai, S.; Li, L. Colloids Surf, B 2005, 43, 143-149. (26) Zhu, A. P.; Chen, T.; Yuan, L. H.; Wu, H.; Lu, P. Carbohydr. Polym. 2006, 66, 274-279. (27) Zhu, A. P.; Fang, N.; Chan-Park, M. B.; Chan, V. Biomaterials 2005, 26, 6873-6879. (28) Zhu, A. P.; Fang, N. Biomacromolecules 2005, 6, 2607-2614. (29) Zhu, A. P.; Chen, T. Colloids Surf., B 2006, 50, 120-125. (30) Zhu, A. P.; Yuan, L. H.; Lu, Y. Colloid Polym. Sci. 2007, 285, 1535-1541. (31) Ma, Z. Y.; Guan, Y. P.; Liu, H. Z. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 3433-3439. (32) Renshaw, P. F.; Owen, C. S.; McLaughlin, A. C.; Frey, T. G.; Leigh, J. S., Jr. Magn. Reson. Med. 1986, 3, 217-225. (33) Furusawa, K.; Nagashima, K.; Anzai, C. Colloid Polym. Sci. 1994, 272, 1104-1110. (34) Sauzedde, F.; Elaissari, A.; Pichot, C. Colloid Polym. Sci. 1999, 277, 1041-1050. (35) Studart, A. R.; Amstad, E.; Gauckler, L. J. Langmuir 2007, 23, 1081-1090. (36) Wan, S. R.; Huang, J. S.; Guo, M.; Zhang, H. K.; Cao, Y. J.; Yan, H. S. J. Biomed. Mater. Res. 2007, 80A, 946-954. (37) Abu-Much, R.; Meridor, U.; Frydman, A.; Gedanken, A. J. Phys. Chem. B 2006, 110, 8194-8203. (38) Di-Marco, M.; Guilbert, I.; Port, M.; Robic, C.; Couvreur, P.; Dubernet, C. Int. J. Pharm. 2007, 331, 197-203. (39) Vincent, B. AdV. Colloid Interface Sci. 1974, 4, 193-277. (40) Napper, D. H. J. Colloid Interface Sci. 1977, 58, 390-407. (41) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (42) Romero-Cano, M. S.; Martin-Rodriguez, A.; de las Nieves, F. J. Langumuir 2001, 17, 3505-3511. (43) Zhu, A. P.; Yuan, L. H.; Liao T. Q. Int. J. Pharm. 2008, 350, 361368.
