Human-like collagen protein-coated magnetic nanoparticles with high magnetic hyperthermia performance and improved biocompatibility
© Liu et al.; licensee Springer. 2015
Received: 25 November 2014
Accepted: 12 January 2015
Published: 31 January 2015
Human-like collagen (HLC)-coated monodispersed superparamagnetic Fe3O4 nanoparticles have been successfully prepared to investigate its effect on heat induction property and cell toxicity. After coating of HLC, the sample shows a faster rate of temperature increase under an alternating magnetic field although it has a reduced saturation magnetization. This is most probably a result of the effective heat conduction and good colloid stability due to the high charge of HLC on the surface. In addition, compared with Fe3O4 nanoparticles before coating with HLC, HLC-coated Fe3O4 nanoparticles do not induce notable cytotoxic effect at higher concentration which indicates that HLC-coated Fe3O4 nanoparticles has improved biocompatibility. Our results clearly show that Fe3O4 nanoparticles after coating with HLC not only possess effective heat induction for cancer treatment but also have improved biocompatibility for biomedicine applications.
KeywordsHuman-like collagen Magnetic nanoparticles Magnetic hyperthermia Biocompatibility
Recently, magnetic nanoparticles (NPs) have been applied in many biomedicine fields because of their appealing magnetic properties [1-4]. In particular, magnetic NPs could be used as magnetic hyperthermia agents for the treatment of cancer [5,6]. Until now, superparamagnetic Fe3O4 NPs, the only clinically approved metal NPs, are widely used in these bio-related investigations because of their superparamagnetism, large specific surface area, and enhanced reactivity . Although superparamagnetic NPs offer rapid growth and therapeutic benefits, at the same time, there are risks and concerns related with their exposure to cells. Therefore, there is a considerable need to address biocompatibility and biosafety concerns associated with their usage in a variety of applications.
Several studies have been reported that the mechanism of toxicity induced from NPs is mainly because of the generation of reactive oxygen species (ROS), which could indirectly damage DNA, proteins, and lipids and results in cell death [8-10]. To date, significant improvements to the cell toxicity of superparamagnetic NPs have been made. Various biocompatible surfactants or polymers have been applied for surface modification of superparamagnetic NPs to reduce its toxicity. For example, albumin-derived superparamagnetic NPs did not result in cell death compared with uncoated superparamagnetic NPs [11,12]. Uncoated superparamagnetic NPs induced greater toxicity compared to that of NPs after coating with biocompatible polyvinyl alcohol (PVA) . Citrate-coated superparamagnetic NPs have been shown to lead to cellular oxidative stress in rat macrophages without causing any toxicity effects . However, certain sizes of superparamagnetic NPs possess low performance of magnetic hyperthermia after being optimized by surface coating for its biocompatible and soluble. This could be due to the low saturation magnetization and reduced contribution of Brown relaxation on heat after surface modification . Moreover, nanoparticles are not well dispersed after being coated with the polymer, which also influences the performance of magnetic hyperthermia. It is critical to optimize magnetic NPs for high heat transfer efficiency and at the same time possess good biocompatibility and colloidal stability in aqueous solution. Hence, it is imperative to design superparamagnetic NPs with specifically tailored surface to meet the demands of the rapidly proliferating field of magnetic hyperthermia application.
Recently, much effort has been expended to design biomaterials which can offer biocompatibility, especially the engineered human-like collagen (HLC). HLC is a special protein and is expressed by recombinant Escherichia coli with a modified cDNA fragment transcribed from the mRNA coding for human collagen [15,16]. Different from animal-derived collagen, HLC has excellent biocompatibility and can easily dissolve in aqueous solutions [17,18]. However, to the best of the authors’ knowledge, there are few cases reported about the HLC modified superparamagnetic NPs to be used as magnetic hyperthermia agents for cancer treatment. In the present study, highly monodispersed Fe3O4 NPs are employed to study the effect of HLC-coated Fe3O4 NPs on both the efficiency of magnetic hyperthermia and cell toxicity. This paper represents one of the first attempts at investigating the effect of HLC-coated superparamagnetic NPs on the magnetic hyperthermia performance and its biocompatibility.
Hexane (J.T. Baker, 99.0%; Avantor Performance Materials, Inc., Center Valley, PA, USA) and absolute ethanol were used as received. Ethyl acetate (99.5%) was purchased from Fluka (St. Louis, MO, USA). Iron (III) acetylacetonate (Fe(acac)3; 97.9%), benzyl ether (99%), oleic acid (90%), acetonitrile (≥99.0%), and sodium periodate (≥99.8%) were purchased from Aldrich Chemical Co. (St. Louis, MO, USA ).
Preparation of highly monodispersed Fe3O4 NPs
As described previously [5,19], high-quality Fe3O4 NPs were synthesized by high-temperature thermal decomposition method. Under a flow of nitrogen, Fe(acac)3 (6 mmol), oleic acid (20 mmol), and benzyl ether (50 mL) were mixed by magnetic stirring. The mixture was first heated to 165°C for 30 min and then heated to 280°C for refluxing under a nitrogen atmosphere for another 30 min. Finally, the mixture was allowed to cool down to room temperature naturally. Ethanol (40 mL) was then added to the mixture under ambient conditions. The product was separated by centrifugation and re-dispersed into hexane.
Transfer of Fe3O4 NPs into water
The transfer of Fe3O4 NPs from hexane to water was through a simple method, which is by oxidation of oleic acid [20,21]. Firstly, the mixture of ethyl acetate and acetonitrile at 1:1 volume ratio was added into the hexane containing as-synthesized Fe3O4 NPs (10 mg). Sodium periodate aqueous solution (40 mg per 1.5 mL) as oxidative agent was then added under vortex mixture. After 2 h, the upper hexane layer was discarded and the aqueous solution at the bottom was magnetically separated. After repeated washing with distilled water (three times), the obtained Fe3O4 NPs were re-dispersed in water.
Coating of HLC on the surface of hydrophilic Fe3O4 NPs
Coating of HLC on the surface of hydrophilic Fe3O4 NPs was performed by using standard (1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride)/N-Hydroxysuccinimide (EDC/NHS).
The phase of as-synthesized Fe3O4 NPs was characterized by X-ray powder diffraction on a Bruker D8 Advanced Diffractometer System (Bruker AXS, Inc., Madison, WI, USA) equipped with Cu/Kα radiation in the 2θ range from 20° to 80° (λ = 1.5418 Å). The size and morphology of samples were characterized using a JEOL 100CX transmission electron microscope (TEM; JEOL Ltd., Akishima-shi, Japan). The mean particle size was obtained from TEM images by counting more than 100 particles. The structure of the particles was characterized using a high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) on a JEOL100CX TEM. Dynamic light scattering (DLS) measurements were performed in a Malvern Zetasizer Nano-ZS device (Malvern, WR, UK) to determine the hydrodynamic size of Fe3O4 NPs before and after coating HLC in a colloidal suspension. The zeta-potential of the suspensions was measured at 25°C. UV-vis absorption spectra were taken using a Shimadzu UV-1601 UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). Magnetic properties of the samples were characterized by a LakeShore Model 7407 vibrating sample magnetometer (VSM; Lake Shore Cryotonics Inc., Wersterville, OH, USA).
NIH3T3 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in 5% CO2 atmosphere at 37°C. Cells were seeded into a 96-well plate at a concentration of 8,500 cells/well. After 24 h, 20 μL magnetic suspensions with various Fe concentrations (25 to 250 μg/mL) were added to each well for co-incubation for 24 h. And then, CCK-8 (10 μL) was added to each well and the samples in the 96-well plate were further incubated for a further 4 h before the absorbance readings, which were conducted at 450 nm using FluoStar Optima microplate reader (LUOstar OPTIMA, BMG Labtech GmbH, Germany).
Results and discussion
Measured lattice spacing
Superparamagnetic Fe3O4 NPs were coated with biocompatible HLC to investigate their magnetic hyperthermia performance and cell toxicity. The results show that the HLC-coated Fe3O4 NPs had a faster rate of temperature rise in magnetic hyperthermia, which result from the higher heat conduction and larger Brownian contribution to heat transfer. Moreover, the biocompatibility was improved after coating with HLC. Surface functionalization of Fe3O4 NPs with biocompatible HLC gave improved stability, heating efficacy, and reduced toxicity towards normal cells, thereby enhancing the potential of magnetic hyperthermia in cancer treatment.
This study was financially supported by the National Natural Science Foundation of China (21276210, 21376190 and 21376192), the National High Technology Research and Development Program of China (863 Program, 2014AA022108), and the Research Fund for the Doctoral Program of Higher Education China (Grant No. 20126101110017). The authors thank Yong Teck (Department of Materials Science and Engineering, Faculty of Engineering, National University of Singapore) for manuscript revision.
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