Synthesis of porous iron oxide microspheres by a double hydrophilic block copolymer

Abstract

Porous microspheres of iron oxide (Fe₂O₃) are synthesized using a double hydrophilic block copolymer of poly(ethylene oxide)-block-poly(acrylic acid) (PEO-b-PAA). The PAA block with negative charge strongly interacts with ferric ions. The assembly of primary nanoparticles at elevated temperature forms the porous microsphere of Fe₂O₃. The MTT assay of the microspheres based on HepG2 cell indicates good biocompatibility. The thorough porosity of microspheres can accommodate a large amount of anticancer drug cisplatin.

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Magnetic particles provide a valuable platform in various biomedical applications including the separation of biological entities,¹ MRI contrast enhancement,² drug delivery,³ and magnetic storage media.⁴ After the experimental investigations of magnetic materials for hyperthermia by Gilchrist et al.,⁵ more attention has been paid to the synthesis of new magnetic particles for thermotherapy for cancer treatment. A number of studies have demonstrated the anticancer efficacy of magnetic hyperthermia.⁶ Magnetic particles exposed to a radio frequency absorb energy from the applied field and dissipate this energy through magnetic relaxation effects, thus results in raising temperature which kills cancer cells.⁷ There have been numerous reports describing a variety of schemes using different types of magnetic materials, different field strengths and frequencies, and different methods of encapsulation and delivery of different theranostic agents.⁸ Magnetic hyperthermia is currently in clinical trials for different cancer cells.⁹

Magnetic property, biocompatibility, biodegradability, and chemical stability of magnetic particles in the physiological environment make them suitable for biomedical applications. Recently, material scientists have demonstrated considerable interest in the synthesis of magnetic particles for multimodality diagnosis and therapy purposes.^10–15 Micelles of stimuli responsive block copolymer were used to synthesize multifunctional magnetic nanoparticles.¹⁰ Xuan et al. synthesized a SiO₂ layer-coated Fe₃O₄ mesoporous spheres and used them to deliver both hydrophobic and hydrophilic drugs.¹¹ Inhalable magnetic nanoparticle for targeted hyperthermia in liver tumor was reported by Sadukha et al.¹² High temperature decomposition of organo-metallic precursors such as Fe(CO)₅ in organic solvents offers improved control over the size and shape of magnetic nanoparticles.¹³ Polyacrylic acid was encapsulated into the inner cavity of Fe₂O₃ hollow spheres by a vacuum casting route and photo-initiated polymerization. Polymer functionalized microspheres of magnetic nanoparticles showed dual responses of magnetic field and pH change.¹⁴

Regarding more practical biological applications, there have been several reports about in vitro studies of biological cells internalizing magnetic nanoparticles submitted to a radiofrequency magnetic field. For example, Fortin et al. reported a cellular death increase by pure hyperthermic effect.¹⁶ Other groups reported cellular toxicity without any measured temperature increase, but with a high cellular uptake efficiency thank to the use of antibodies.¹⁷ Another strategy consists in submitting cells to magnetic drug-loaded nanovectors, which liberate an anticancer drug (e.g., doxorubicin) to the cells under local temperature increase.^18,19

As far as the application in biological fields is concerned, the porous nanostructure with large surface area, good dispersion in aqueous media are of particular importance because they can act as promising drug delivery carriers and provide efficient signal owing to their porous interior structure and sufficient magnetic saturation. Preparation of biocompatible porous magnetic particles with uniform size still remains a challenge. Here we report one-pot synthesis of highly biocompatible porous Fe₂O₃ microspheres with assistance of double hydrophilic block copolymer, poly(ethylene-block-acrylic acid) (PEO-b-PAA). The PAA block with negative charge strongly interacts with ferric ions.²⁰ Nucleation of Fe₂O₃ nanoparticles is expected to be promoted by carboxylic acid groups on the PAA segments in the presence of the urea at elevated temperature in aqueous solution. The assembly of primary nanoparticles forms the microspheres of Fe₂O₃. The highly biocompatible and highly porous microspheres can accommodate large amount of anticancer drug cisplatin and can be used as a nanocarrier for several theranostic molecules.

In the experimental, FeCl₃ (25 mg) was added to a PEO-b-PAA solution (40 mL, 0.2 g L⁻¹). Urea (60 mg) was added into the solution and then magnetically stirred for 1 hour. The resultant yellow solution was kept at 100 °C in autoclave for 5 hours. The precipitation was collected and washed thoroughly with distilled water and dried at 50 °C in vacuum oven. As-prepared samples were calcined at various temperatures from 200 °C to 500 °C for 5 hours.

In the present approach, magnetic Fe₂O₃ microspheres were synthesized by using one-pot hydrothermal method. PEO-b-PAA double hydrophilic block copolymer used in this study was served as structure directing agent and chelating ligand. The highly reactive PAA block can chelate the Fe³⁺ ions by ionic interaction. The Fe³⁺ ions were used as an iron source for the formation of Fe₂O₃. PEO-b-PAA is a typical double hydrophilic block copolymer containing a strongly interactive carboxyl-functionalized block, i.e., the PAA block, which has been previously used in the controlled mineralization/synthesis of several compositions, such as calcium carbonate,^21–24 calcium phosphate,²⁵ ceria,²⁶ and magnetic nanoparticles.^27,28 The present approach provides the platform for the formation of primary nanoparticles and undergoes simultaneous assembly to form the monodispersed porous microspheres. The soft-chains of block copolymer binds onto the nanoparticle surface provide mobility to the primary nanoparticles, which would allow for reconstructing the morphologies and structures of nanoparticle assembly to form microspheres.

The average hydrodynamic diameter (D_h) of as-prepared sample was determined by using dynamic light scattering measurement. D_h was found to be 250 nm as shown in Fig. 1a. The obtained Fe₂O₃ particles were easily dispersed in water. Fig. 1b shows the SEM image of the as-prepared monodispersed Fe₂O₃ microspheres. These particles are spherical and remarkably uniform with an average size about 200 nm. The as-prepared microspheres show bumpy surface which seems aggregation of several small nanoparticles, as was clearly observed by SEM image (Fig. 1b).

Fig. 1 (a) Hydrodynamic diameter of as-prepared Fe₂O₃ microspheres and (b–f) SEM image of (b) as-prepared and (c–f) calcined samples at (c) 200 °C, (d) 300 °C, (e) 400 °C, and (f) 500 °C.

In general, hydrophilic PEO stabilize the colloidal particles in aqueous solution in the presence of ions. However, some other interactions of PEO with either ionic or neutral additives have been reported.²⁹ Ferric ions are coordinated with carboxylic acid groups of PAA segments to form the complexes, and then the nucleation of magnetic nanoparticles is promoted by carboxylic acid groups on the PAA segments in the presence of the urea at elevated temperature in aqueous solution. The excess Fe³⁺ ions interact with PEO as it looks like a crown ether. This interaction weakens the hydration of ethylene oxide group,^30,31 which promotes the assembly of preformed primary Fe₂O₃ nanoparticles to form the microspheres.

To check the thermal stability, the as-prepared microspheres were calcined at different temperatures. The calcination did not destroy the morphology at all. Fig. 1 shows the SEM images of the microspheres calcined at different temperatures. Quantitative analysis of PEO-b-PAA contained in the as-prepared Fe₂O₃ microspheres was checked by thermogravimetric (TG) analysis, as shown in Fig. S1.† Large weight loss (15 wt%) at around 300 °C corresponds to desorption of water molecules and decomposition of polymers during calcination process. The lowest calcination temperature applied in this study was 200 °C, but the calcination time was set for 5 hours which was enough to completely remove the polymer at elevated temperature.

Crystallinity and phase purity of the obtained microspheres were checked by wide-angle XRD measurement. Fig. S2† shows wide-angle XRD patterns for the samples calcined at different temperatures. The as-prepared microspheres were amorphous, but the crystallinity was gradually enhanced with the increase of calcination temperature. After calcination at 200 °C, very weak and broad peaks assignable to γ-Fe₂O₃ phase were observed at 2θ = 35° and 63°, as indicated by asterisks (*). When the calcination temperature was increased up to 300 °C, the γ-phase started transferring to α-phase. Above 400 °C, the peaks derived from α-Fe₂O₃ phase were developed. All the peaks could be readily indexed as the α-Fe₂O₃ phase (JCPDS 00-033-0664). With the increase of calcination temperature, the peaks were sharper and HWHM (half width at half maximum) of the peaks was narrower, indicating gradual growth of the crystals. From SEM image of the sample calcined at 600 °C (Fig. 1f), the particle surface became smooth.

The surface areas of the samples calcined at different temperatures were investigated by N₂ adsorption–desorption isotherms (Fig. S3†). The sample calcined at 200 °C showed the highest surface area of 120 m² g⁻¹. Above 300 °C, the surface areas were gradually decreased. As mentioned above, with the increase of calcination temperature, the large crystals were formed and they were more densely assembled each other, thereby decreasing the accessible surface areas. The surface area of as-prepared sample with amorphous phase was lower than that of the sample calcined at 200 °C, due to the presence of the remaining polymers.

Room-temperature magnetization curve of the sample calcined at 200 °C was measured as a function of the applied magnetic field (Fig. S4†). The applied magnetic field was changed between +12 000 and −12 000 Oe at room temperature. Under a large external field, the magnetization of the particles aligns with the field direction. The magnetization was not completely saturated at magnetic field of 12 000 Oe. No hysteresis was observed in the magnetization curve (the coercive forces (H_c) and residual magnetization (M_r) were almost zero), which is typical of superparamagnetic behavior. Generally, the γ-Fe₂O₃ phase which is the main phase in the sample is ferromagnetic. But, very small ferromagnetic grains show superparamagnetic behavior; that is, each grain does not show a ferromagnetic state because of the thermal fluctuation of the magnetic moment at room temperature. The magnetization value of the sample calcined at 600 °C, measured at magnetic field of 12 000 Oe, was small (only 0.5 emu g⁻¹). This is an evidence of the completion of the phase transfer of γ-Fe₂O₃ to α-Fe₂O₃.

The porous structure facilitates the immobilization and subsequent encapsulation of substances in medical use. There has been increased interest in inorganic porous materials as drug carriers. Therefore, we investigated incorporation of anticancer drug, cisplatin, into porous Fe₂O₃ microspheres. The cisplatin with Pt content can be used as probe molecules, because the heavy element such as Pt can be easily detected by elemental mapping. The location of the loaded cisplatin was confirmed by elemental mapping on TEM observation, as shown in Fig. 2. It was clearly seen that Pt content (derived from cisplatin) was uniformly distributed over the entire area of microspheres. From this result, it was proved that the cisplatin molecules were introduced into the inner part of microspheres, meaning the porous structures inside the microspheres is also effective for the adsorption of cisplatin molecules. The loading capacity of the sample calcined at 200 °C was found to be 35% w/w which is higher than previously reported value of similar system.³²

Fig. 2 (a) Bright-field TEM image of Fe₂O₃ microspheres calcined at 200 °C and (b) HAADF-STEM image of one Fe₂O₃ microsphere calcined at 200 °C (c) elemental mapping image after loading cisplatin.

The interior structure of microspheres was further visualized by TEM observation. The typical TEM image of the sample calcined at 200 °C is shown in Fig. 2a. All the microspheres were porous and composed of small particles rather than formed by homogeneous Fe₂O₃. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to assess cellular viability of the materials on liver cancer cell line, HepG2. The result in Fig. 3a shows the cell viability exceeded 95% even when the particle concentration was as high as 1000 μg mL⁻¹, indicating a good biocompatibility in vitro. This clearly ensures their safe application for drug delivery. Thus, compared to many other inorganic materials, our porous Fe₂O₃ microspheres are exceedingly suitable because of its good biocompatible and biodegradable properties.

Fig. 3 (a) Cell viability of HepG2 cells after incubation for 24 h in 37 °C. (b) Images of liver cancer cells HepG2 (b-1) before and (b-2) after uptake of Fe₂O₃ microspheres.

The images of liver cancer cells HepG2 before and after uptake of the porous Fe₂O₃ microspheres were taken and shown in Fig. 3b. The cells were stained with nuclear fast red before the microscopic observation, and they all displayed elongated shape with intact structure (Fig. 3b-1). For those cells treated with iron oxide samples, the cells were stained in order to observe the Fe₂O₃ microspheres. In contrast to Fig. 3b-1, there were many blue spots representing the existence of iron oxide samples (Fig. 3b-2). In addition, the image in the inset of Fig. 3b-2 clearly indicated that the Fe₂O₃ microspheres were indeed internalized by the HepG2 cells and aggregated in the cytoplasm of the cells. In the experimental, we washed out the cells very carefully before the microscopy observation. Therefore, we believe that the particles were internalized inside the cells, not attached on the cell surface.

In conclusion, we successfully developed a new hydrothermal method with double hydrophilic block copolymer to synthesize highly uniform, biocompatible, magnetic, and porous Fe₂O₃ microspheres with high surface area. High loading capacity makes the material as a promising candidate for magnetically guided nanocarriers. Our approach is widely applicable to other porous metal oxide microspheres with various properties, by selecting inorganic sources.

Acknowledgements

The research was supported by the National Science Council of Taiwan (101-2628-E-002-015-MY3), National Health Research Institute (NHRI) of Taiwan (ME-102-PP-14), National Taiwan University (102R7842 and 102R7740) and Center of Strategic Materials Alliance for Research and Technology (SMART Center), National Taiwan University (102R104100).

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and characterization of the synthesized porous Fe₂O₃ microspheres. See DOI: 10.1039/c3ra47490a

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