Biomagnetic of Apatite-Coated Cobalt Ferrite: A Core–Shell Particle for Protein Adsorption and pH-Controlled Release
© Tang et al. 2010
Received: 22 June 2010
Accepted: 14 August 2010
Published: 31 August 2010
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© Tang et al. 2010
Received: 22 June 2010
Accepted: 14 August 2010
Published: 31 August 2010
Magnetic nanoparticle composite with a cobalt ferrite (CoFe2O4, (CF)) core and an apatite (Ap) coating was synthesized using a biomineralization process in which a modified simulated body fluid (1.5SBF) solution is the source of the calcium phosphate for the apatite formation. The core–shell structure formed after the citric acid–stabilized cobalt ferrite (CFCA) particles were incubated in the 1.5 SBF solution for 1 week. The mean particle size of CFCA-Ap is about 750 nm. A saturation magnetization of 15.56 emug-1 and a coercivity of 1808.5 Oe were observed for the CFCA-Ap obtained. Bovine serum albumin (BSA) was used as the model protein to study the adsorption and release of the proteins by the CFCA-Ap particles. The protein adsorption by the CFCA-Ap particles followed a more typical Freundlich than Langmuir adsorption isotherm. The BSA release as a function of time became less rapid as the CFCA-Ap particles were immersed in higher pH solution, thus indicating that the BSA release is dependent on the local pH.
The targeting of biomolecules (e.g., drug, DNA, or protein) using delivery vehicles that are biocompatible with the human body is an area of great interest. To combine into one vehicle, two types of materials, a magnetic material that has the magnetic property needed for the targeting and a biopolymer that possess a biocompatibility with the human body, would be of a great advantage to the treatment of human illnesses [1–6]. Magnetic composites can also be used in magnetic bioseparation [7–12], drug delivery [13–16], and hyperthermia [17–22] applications. The particles in all of these applications should have a core/shell structure, with the core being the magnetic material. The core must be pre-coated with a shell to insure their stability, biodegradability, and non-toxicity in the physiological medium [23–27]. These shells are often made with biopolymers. The shell can also be made with inert materials such as calcium phosphate of apatite. Hydroxyapatite has an excellent biocompatibility with the human body and degrades slowly in physiological medium (pH ~ 7.4) [28–31]. Apatite is also an ideal material for the surface coating of the core since its surface is porous that allows for the adsorption of biological molecules. This is very important since the purification and separation of protein and enzymes are achieved by attaching (functionalizing) chemical radicals that have an affinity for these biomolecules for the nanoparticles.
In this study, we are interested in vehicles (magnetic nanocomposites) that can be guided to their targets by the action of a magnetic field. The targeting of the biomolecules would lessen the possible toxic effects the biomolecules may have on the human body when too much of the biomolecules are introduced into the human body. The toxic effects would also be lessened by the coating. The coating may however lead to changes in the composition, size, morphology, and surface chemistry of the magnetic particles. These changes in turn may lead to changes in the magnetic behavior of core material in vivo. The response of the magnetic core to an external field should be the same after the coating.
Fe3O4 (magnetite) was the magnetic material used [1–6, 15–20] in the initial applications of magnetic nanoparticles. Another magnetic material which also has been used is CoFe2O4 (cobalt ferrite) [32–34]. Cobalt ferrite has a higher magneto-crystalline anisotropy that results in it having a higher coercivity field, Hc (the field at which the magnetization is zero). Importantly, it possesses good chemical stability. This is needed since the functionalization of magnetic nanoparticle by the carboxylic groups is performed in a mild acidic solution. In this study, cobalt ferrite was therefore chosen to be the core material.
Composites of magnetic particles and calcium phosphate have been studied already. Wu et al.  showed that the calcium phosphate of hydroxyapatite-based magnetic nanoparticles has good biocompatibility with the human body and that it does not elicit any cytotoxicity. Pareta et al.  showed that in the presence of bovine serum albumin (BSA) protein, the osteoblast density of magnetic particles coated with calcium phosphate increased significantly after 1 day of immersion. The BSA is one of the most abundant proteins found in blood plasma. Its high content of carboxyl (–COO-) and amino (–NH3+) groups makes it a useful tool in the study of the adsorption of proteins.
We report here a method for synthesizing apatite-coated citric acid–stabilized cobalt ferrite particles (CFCA-Ap) that have a core–shell structure. Its structure, morphology, chemical stability, and magnetic properties of products have been investigated. BSA was used as a model protein to study the adsorption and sustained release of proteins from the CFCA-Ap particles in simulated protein delivery experiments.
Apatite (Ap)-coated citric acid (CA) stabilizes cobalt ferrite (CoFe2O4) (CFCA-Ap) particles that were fabricated in a two-step process. (1) Carboxylic groups from citric acid (COO-) were used to stabilize the surface of the magnetic cobalt ferrite (CF). This was done by immersing the CF in 0.001 M citric acid. At this point, citric acid–stabilized cobalt ferrite (CFCA) particles were formed (CF suspension in aqueous solution). (2) For the biomimetic process, the CFCA particles were then immersed in a simulated body fluid (SBF) solution. The anionic surface of CFCA particles accelerated the chelating of the calcium ions present in the SBF solution onto the surface of the CFCA. These then react with the phosphate ions (also in the SBF) to form the apatite layer on the cobalt ferrite surface. These became the apatite-coated critic acid–stabilized cobalt ferrite (CFCA-Ap) core–shell particles. The individual steps in the fabrication process are as follows:
The cobalt ferrite nanoparticles were prepared using NaOH, Fe(NO3)3 · 9H2O and Co(NO3)2 · 6H2O obtained from UNIVAR (Australia). The sodium dodecyl sulfate (SDS), Na2SO4, and ethylene glycol (EG) were obtained from Fluka (Switzerland). The ratio of Fe to Co was kept at 2:1 mol%. Each chemical constituent was dissolved in 25 ml de-ionized water. These solutions were then mixed together. This was then added to a solution of 3 M ethylene glycol (5 ml) and 0.001 M Na2SO4 (3 ml). The pH of the resulting CoFe2O4 precipitate was adjusted by adding NaOH under constant stirring conditions. The temperature at which the reaction occurred was kept at 60°C throughout. After adding the NaOH to the solution, the formation of black particles of cobalt ferrite could be seen. After the completion of the reaction, the solution was continuously stirred for another 30 min. The cobalt ferrite powder was obtained by centrifuge and freeze drying.
The SBF is a fluid having an ionic composition very similar to that of human plasma [37, 38]. The SBF solution we used is one of the more extensively used solution. It consists of the following chemicals: NaCl (136.8 mM), NaHCO3 (4.2 mM), KCl (3.0 mM), K2HPO4 (1.0 mM), MgCl2 · 6H2O (1.5 mM), CaCl2 (2.5 mM) and Na2SO4 (0.5 mM). These chemicals are mixed together with the pH adjusted to 7.4. The SBF is replaced every 3 days to avoid any changes in the cationic concentration that may occur due to the degradation of the sample.
The crystal structures of the powders were determined by powder X-ray diffraction (XRD) (Bruker diffractometer, Model D8 Advance) using the Cu Kα radiation and operating at 40 kV with 40 mA current. The XRD patterns were scanned from 2θ = 20°–60° at a scanning speed of 1 s per step with an increment of 0.037° per step. For the FT-IR absorption measurements, the powders were mixed with KBr and pressed into pellets using a pressure of 10 tons for 1 min. The pellets were analyzed using a FT-IR spectrophotometer (Spectrum GX, Perkin Elmer) that performed 16 scans over the range 370–4,000 cm-1. The magnetic properties of CFCA-Ap particles were measured using a room temperature VSM (vibrating sample magnetometer (Lakeshore, Model 4500)). The coercivities (Hc), the remnant magnetizations (Mr), and the saturation magnetizations (Ms) of the samples were measured with the VSM. The room temperature magnetic parameters (Hc, Mr, and Ms) of each sample were determined from the hysteresis loops produced by the VSM. A scanning electron microscope (SEM) (JEOL model JSM-6301F) was used to observe the size and morphology of the samples. An accelerating voltage of 15 kV was used to obtain the SEM images. The formation of apatite crystals on the cobalt ferrite surface was analyzed with an energy dispersive spectroscopy (EDS) (ISIS 300 (Oxford Instruments)). The core–shell particles were also examined by a transmission electron microscope (TEM) (JEOL model JEM-2010). The electron diffraction attachment to the TEM was used to obtain electron diffraction patterns of CFCA-Ap particles. Particle size distribution and zeta-potential measurement of CFCA-Ap were measured with a Zetasizer Nano ZS (Malvern, UK).
Various amounts of BSA (Fluka, Switzerland) were dissolved in phosphate buffer saline (PBS) with a pH of 7.4 to obtain final concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg/ml. Twenty-five milligrams of the CFCA-Ap particles was mixed for 4 h with 5 ml of each BSA concentration using a mildly magnetic stirrer. The temperature was kept at 37°C throughout the experiment. At the conclusion of this mixing, the protein-loaded CFCA-Ap powders were removed from the liquid by a permanent magnet. The supernatants were kept for later use.
The in vitro study of BSA protein release from the core–shell CFCA-Ap particles was performed as follows (the concentration of 0.6 mg/ml of BSA protein loaded on CFCA-Ap particles was chosen for study): the BSA-loaded CFCA-Ap particles were placed in 15 ml of phosphate buffer (having pH = 4.0, 6.5 and 7.4) (Fisher Scientific, UK) with the temperature being 37°C. The release of the BSA protein from CFCA-Ap was done by using a shaking bath (SKAKER, Sk-300). The length of time that the BSA protein loaded on CFCA-Aps kept in the PBS buffer was varied (the different time periods used are 0.5, 1, 2, 3, 4, 5, 10, 20, and 30 h). At the end of each period, 3 ml of the supernatant was taken out. The concentration of the BSA protein in each of the 3 ml volumes was determined by using a UV–Vis spectrophotometer (Jenway, model 6405) operating at a wavelength of 280 nm. After each measurement, the supernatant was returned to the system. Assuming that the measurement time is very short compared to the time needed for the BSA protein release, the influence of this return on the release profile is expected to be negligible. The protein content in supernatant is again determined by comparing the measured absorbance to a standard curve for the BSA adsorption, represented by the following equation: C i = 0.5709A i + 0.0654; R2 = 0.9931, where C i is the BSA protein concentration and A i is the UV–Vis absorbance at a wave length of 280 nm.
The BSA in the burst release when the pH is 4.0 does not only originate from the diffusion of proteins entrapped near the surface but also from within the CFCA particles since the calcium phosphate in the apatite coating on the particles surface dissolves when the environment is acidic. Both phenomena occur simultaneously. This leads to the release of the BSA from the CFCA particles to be very rapid. When the pH is higher, i.e., at pH of 6.5 and 7.4, the release rate is lower since only the proteins trapped at the surface are released. The proteins entrapped within the CFCA-Ap can only be released when they diffuse through the apatite layer and reach the surface. The low permeability of BSA through the porous apatite layer will lead to a low release.
Isotherm constants for BSA adsorption onto apatite (Ap)-coated citric acid–stabilized cobalt ferrite (CFCA) (CFCA-Ap) particles
Langmuir isotherm parameter
Freundlich isotherm parameter
From the release profile of BSA protein from CFCA-Ap particles, the results (shown in Figure 8) show that the release depends on the pH. Two behaviors are observed: a bust release in the first 5 h especially when the release is occurring in an acidic media (pH 4.0 and 6.5), or a gradual release occurring after 5 h when the pH is at a physiological level of 7.4. It thus appears that the release of BSA from CFCA-Ap particle is controlled by the pH of the surrounding medium. Why this occurs, we need to consider the manner of the release of BSA from apatite [42, 43]. First of all, there are positively charged Ca2+ ions and negatively charged PO43- ions on the CFCA-Ap particles. The BSA contains negatively charged carboxyl (–COO-) and positively charged amino (–NH3+) as side groups. The interaction between the opposite charges on the apatite with the BSA inhibits the release of the protein until the calcium phosphate of apatite is dissolved.
This indicates that the release rate of BSA is pH sensitive and that the release constant k decreases with increasing pH. The higher rate of release in acidic environment results from abrupt morphological changes caused by dissolution of the calcium phosphate of apatite. This implies that the BSA release is mainly controlled by the particles dissolution which in turn leads to the release of tightly bound BSA.
Release characteristic parameters of release profiles of BSA adsorption on apatite (Ap)-coated citric acid–stabilized cobalt ferrite (CFCA) (CFCA-Ap) particles
Y = kt n ; y = M t /M0
Y = φb(1 - exp(-kbt)); y = M t /M0
K (h -1 )
k b (h -1 )
BSA-loaded CFCA-Ap particles were synthesized through the biomineralization of Co-ferrite particles in a 1.5SBF solution after which BSA loaded on. XRD and FT-IR spectra indicated that the coating on the CFCA-Ap particles consisted of apatite mineral. The core–shell structure of the CFCA-Ap particles was observed by TEM. The sizes of the particles were in the range of 400–1,200 nm. The CFCA-HAp particles have a good capacity for adsorption of BSA. The adsorption is better described by a Freundlich isotherm than by a Langmuir isotherm. It is found that the release of BSA by the CFCA-Ap particles is pH dependent. The release of the BSA from the CFCA-Ap particles is greatly accelerated when the pH of the medium is lowered. These results suggest that CFCA-Ap particles can be useful for the sustained release of protein or other biomolecules (e.g., DNA), in an acidic media. This would be especially important if the acidity is due to a bacterial infection or inflammation.
The authors would like to thank Thailand Center of Excellence in Physics (ThEP) for support.
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