Chemical modifications of Superparamagnetic Iron Oxide Nanoparticles (SPION) surfaces by attachment of functional groups and further covalent coupling with ...
Mat. Res. Soc. Symp. Proc. Vol. 704 © 2002 Materials Research Society
Surface modification of superparamagnetic nanoparticles for in-vivo bio-medical applications D. K. Kim1, M. Toprak1, M. Mikhailova1, Y. Zhang1, B. Bjelke2, J. Kehr3, M. Muhammed1. 1
Materials Chemistry Division, Royal Institute of Technology, SE-100 44 Stockholm, Sweden MRI-Center, Experimental Unit, Karolinska Institutet, SE-171 76 Stockholm, Sweden 3 Division of Cellular and Molecular Neurochemistry, Karolinska Institutet, SE-171 77 Stockholm, Sweden 2
ABSTRACT Chemical modifications of Superparamagnetic Iron Oxide Nanoparticles (SPION) surfaces by attachment of functional groups and further covalent coupling with biodegradable substances have been studied. Based on computer-assisted chemical equilibrium calculations, several optimum operation conditions for a coprecipitation process of magnetite nanoparticles were predicted. These particles were immobilized by ultra-thin films of PVA, Dextran, Dextrin, PEG and MPEG to obtain a biocompatible particle surface for further functionalization purposes. The effect of surface modification of the superparamagnetic nanoparticles in terms of chemical and physical properties of the samples was investigated with several techniques, including microelectrophoresis measurement. The feasibility of using SPION in biomedical applications was investigated by in-vivo treatment in rat brains. INTRODUCTION Application of SPION for hyperthermia of biological tissue has been known in principle for more than four decades [1]. Especially, for MR imaging purposes, imaging agents are required to increase conspicuity of adjacent internal organs and tissue. Moreover, SPION can be used to monitor extracellular macromolecules both at a single cell level (genes and proteins) and at a network level (intercellular communication) by in-vivo monitoring of particle movement in the living brain tissue. Biomedical applications using SPION require narrow size distribution and surface modifications with biocompatible materials, i.e. nonimmunogenic, nonantigenic, and protein-resistant. To transport this hydrophilic substance in biological membrane system, surface modification of the nanoparticle was necessary to adjust the zeta potential close to zero. The influence of physico-chemical characteristics on the uptake of particles by the mononuclear phagocyte system (MPS) and accumulation in the reticuloendothelial system, comprising mainly of the macrophages of the liver and the spleen has been reported. [2] Also, ferrofluids composed of SPION are not only affected by an inhomogeneous particle size distribution, but also by the surface charge of the particles in the solution. One of the practical methods to stabilize the ferrofluids is through electrostatic stabilization, achieved by the repulsion of equally charged surface. The repulsive force results from creation of an electric double layer around the particles, which is dependent on dispersion of the particles into a polar media, pH, concentration, and ionic strength of the suspension. Specific medical application of these particles also requires a modified surface in order to conjugate with antibodies and/or functional groups[3]. W11.2.1
Figure1. TEM image of naked magnetite prepared by LB technique.
Figure 2. Schematic view of MPEG immobilization on SPION.
SAMPLE PREPARATION AND EXPERIMENTAL METHODS All chemicals used were of reagent grade and used without further purifications. Ferric chloride hexahydrate (FeCl3·6H2O > 99%), ferrous chloride tetrahydrate (FeCl2·4H2O >99%) and starch ((C6H10O5)n >99%) were obtained from Aldrich. Sodium hydroxide (NaOH >99%) and hydrochloric acid (HCl > 37%) were obtained from KEBO. Milli-Q water (18MΩcm) was redeionized (specific conductance < 0.1 s/cm) and deoxygenated by bubbling N2 gas for 1 hr before use. 2g of starch were dissolved in 50 ml of distilled and deionized water. The resulting solution was vigorously stirred and heated to 70°C under a N2 atmosphere. After the starch was thoroughly dissolved, the solution was immediately placed in a 50°C water bath. Subsequently, 1M FeCl3·6H2O, 0.5M FeCl2·4H2O, and 0.4M HCl were dissolved into a starch matrix solution as a source of iron under vigorous stirring. In the same way, 250mL stock solutions of 1 M NaOH were prepared as alkali sources [5]. The magnetite nanoparticles for MPEG immobilization were prepared by controlled coprecipitation method [6]. The resulting particle suspension was then washed several times with analytical grade methanol with the help of external magnet. Then methanol solution was mixed with toluene and heated at 95°C under
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Figure 3. AFM image of MPEG coated magnetite and section analysis result W11.2.2
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Figure 4. Magnetization curves up to 5T of MPEG immobilized SPION.
Figure 5. ZFC at 10 Oe and FC measurement for MPEG immobilized SPION.
nitrogen until half of the solution was evaporated, so that the water was thoroughly removed. A solution of 3-aminopropyltrimethoxy silane (APTMS) was added to the solution. The ferrofluid was stirred and heated under a N2 atmosphere at 160°C for 12hrs. After cooling down until 80°C, MPEG was added to immobilize the surface of the SPION. The dynamic mobility, called the acoustic pressure amplitude (ESA) was generated by colloidal particles in alternating high frequency electric fields, was measured with a Matec Applied Sciences (ESA-9800) system. 250mL of 1 M NaOH were placed in a Teflon sample container. 1mL of iron stock solution was added by using a MICROLAB 500 Digital burette and stabilized for 10 sec before measuring the data. During the titration, the electro-acoustic signal, electrical conductivity and temperature were recorded as a function of pH. The stirring speed was kept at a maximum value. To receive morphological and structural information on the obtained SPION, the samples were analyzed by X-ray diffraction (XRD) measurements, AFM, and transmission electron microscopy (TEM) imaging. The XRD measurements were performed using a Philips PW 1830 diffractometer, and Scherrer’s formula [7] was used for the estimation of the average crystal size, Dc. The TEM images were taken using a JEOL-2000EX microscope. The mean diameter and standard deviation of the size distribution were calculated with assistance from a image analysis program based on a log-normal function [8]. The magnetization of the samples was measured with a 7 T Quantum Design SQUID magnetometer between 5 -300 K. For zero-field cooled (ZFC) experiments, the sample was cooled down and a constant field was applied during the warm up scan. AFM measurements were performed in the tapping mode of a Nanoscope IIIa system (Digital Instruments, USA). A silicon tip was used in the measurements. The MRI recording was performed on a Biospec Avance 47/40 spectrometer (Bruker, Karlsruhe, Germany) at 4.7Tesla. RESULTS AND DISCUSSION MPEG immobilization on SPION The particle size distribution and morphology were examined by TEM analysis. A Langmuir film was prepared in a TEM grid to prepare the monodispersed SPION monolayer. The particles size determined by image analysis of TEM image, Figure 1, is 42 Å with a standard deviation ,σd, =0.1. This result is close to the crystal size calculated from XRD data (40 Å). The X-ray W11.2.3
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Figure 6. In-situ ESA and conductivity measurements of SPION precipitation reaction in polymeric starch matrix.
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Figure 7. Time dependence of ESA and conductivity of SPION after precipitated in polymeric starch matrix.
diffraction patterns show very low crystallinity at the interface. This induces a change in the magnetocrystalline anisotropy [9]. Even the magnetic diameters calculated from the Langevien equation shows smaller diameters than those determined by TEM. When the magnetite nanoparticle is highly crystalline, the interactions between the particles are significantly lager than for low crystallinity. The magnetic size of SPION powder forms and dispersed in a fluid are reported in previous papers [6]. Figure 2 shows schematic view of covalently immobilization of MPEG on the SPION surfaces. Many kinds of biological substance are hydrophilic and water soluble, so they cannot pass through the hydrophobic lipid bilayer membranes. This is because MPEG has uncharged hydrophilic residues and very high surface mobility leading to high steric exclusion. Therefore, it is expected to effectively improve the biocompatibility of the nanoparticles and to possibly prevent accumulation in the reticuloendothelial system or mononuclear phagocyte system. Figure 3 shows an AFM image of MPEG immobilized SPION. Clusters include several magnetite single particles and the cluster size shows around 200nm. The hysteresis loop measurement from 5K to 300K for the MPEG immobilized SPION is shown in Figure 4. As seen in Figure 4, the typical characteristics of superparamagnetic behavior are observed by showing almost immeasurable coercivity and remanence above the blocking temperature. The SPION are ferri- or ferromagnetic single- domain particles that are significantly small the thermal energy is the same order of magnitude as the anisotropy energy barrier. For an assembly of particles, magnetic moment directions are redistributed by thermal fluctuations and the particles exhibit no remanence without an applied magnetic field [ref]. To produce a stable superparamagnetic core, the SPION particles have to be kept at a distance to reduce the influence of their interactions from the surface of each magnetic nanoparticle. In-situ monitoring of SPION coprecipitation reaction in polymeric starch matrix If the applied electrical field is alternating, the resulting pressure field will alternate as well, resulting in a sound. The intensity per unit electrical field, which is related to the acoustophoretic mobility is given by the expression ESA = c × ∆ρ × Φ × µ (ω ) (1)
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Figure 8. MRI analysis of the distribution of starch coated SPION in a living rat brain (one hour after injection)
Figure 9. Starch coated SPION distribution inside the brain tissue analyzed by 3D mapping.
where c is the sound velocity in the dispersing liquid, ∆ρ is the difference in densities between solid and liquid, Φ is the volume fraction of solid in the dispersion and µ is the dynamic mobility. Figure 6 shows the results of ESA and conductivity measurement of iron oxide nanoparticle formation in the presence of a polymeric starch matrix. Each data point means that 1mL of iron source in polymeric starch matrix was added to the reaction chamber and held 10 sec until the system had stabilized. From the in suit reaction monitoring investigations, indirect information is received on the coprecipitation reaction mechanism. At pH=12.8, the precipitation reaction was started. The results show that conductivity was not changed much until the pH=12.55. It dramatically increased at 4 times after the pH
