Monodisperse magnetic nanoparticles (MNPs) were synthesized by thermal decomposition of iron-oleate and functionalized with silanes bearing various functional groups such as amino group (NH2), short-chain poly(ethylene glycol) (PEG), and carboxylic group (COOH). Then, silanes-functionalized magnetic nanoparticles (silanes-MNPs) were incubated in cell culture medium plus fetal calf serum to investigate the effects of proteins from culture medium on surface property of MNPs. Zeta potential measurements showed that although surface charges of silanes-MNPs were different, they exhibited negative charges at neutral pH and approximate isoelectric points after they were incubated in cell culture medium. The reason was that silanes-MNPs could easily adsorb proteins from culture medium via non-covalent binding, resulting in the formation of protein-silanes-MNPs conjugates. Moreover, silanes-MNPs with various functional groups had different adsorption capacity to proteins, as confirmed by Coomassie blue fast staining method. The in vitro cell experiments showed that protein-silanes-MNPs had higher cellular uptake by cancer cells than silanes-MNPs.
KeywordsMagnetic nanoparticles Culture medium Protein Surface property Cellular uptake
Magnetic nanoparticles have been identified as potential candidates for biological applications including magnetic separation , magnetic resonance image (MRI) [2–4], magnetic hyperthermia [5, 6], and so on. As a result of this drive to integrate MNPs into biological systems, a lot of research has focused on the interactions between MNPs and cancer cells. In terms of specifically biological applications, MNPs are always functionalized with interesting molecules such as PEG [7–10] and folic acid [11, 12], suggesting that surface chemistry of MNPs play an important role in relative topics. It is well known that the in vitro investigations on the interactions between MNPs and cancer are usually carried out in various cell culture mediums such as DMEM and RPMI-1640. At the same time, cell culture medium is a complex system and contains a lot of proteins, which can be adsorbed by MNPs. Thereby, it could be predicted that MNPs may exhibit novel surface properties due to the adsorption of proteins once MNPs are dispersed in cell culture medium. The change of surface properties can inevitably influence the interactions between MNPs and cancer cells because the surface is in direct contact with cells. Unfortunately, it had always been ignored until some research reported recently. Chithrani et al.  found that Au nanoparticles could only be well dispersed in DEME cell culture medium plus 10% serum. Similarly, Casey et al.  reported that single-walled carbon nanotubes could be well dispersed in medium (F12 K) with 5% (v/v) fetal bovine serum (FBS), resulting from interaction of both components of F12 K and FBS with carbon nanotubes likely through a physisorption. Zhu et al.  further demonstrated that the formation of peptone-MWNTs conjugates by non-covalent binding due to the adsorption of peptone in proteose peptone yeast extract medium onto the surface of multi-walled carbon nanotubes (MWNTs) stimulated growth of cells. In our previous study, we reported that fetal calf serum in RPMI-1640 cell culture medium played a great role in the stability of 2, 3-dimercaptosuccinnic acid (DMSA)-coated MNPs under biological conditions, as well as their surface property and intracellular uptake , which is attributed to the adsorption of fetal calf serum onto the surface of MNPs.
In this study, monodisperse MNPs (Fe3O4) were synthesized by iron-oleate thermal decomposition method and functionalized with silanes bearing different functional groups such as NH2, short-chain PEG, and COOH. Despite the distinct surface properties, all of these silanes-MNPs exhibited negative surface charges at neutral pH and approximate isoelectric point (IEP) when incubating in cell culture medium (RPMI-1640 plus 10% fetal calf serum (FCS)). This phenomenon was attributed to the adsorption of proteins from fetal calf serum onto the surface of silanes-MNPs and the formation of protein-silanes-MNPs conjugates. Moreover, the different adsorption capacities of silanes-MNPs to proteins were determined by Coomassie blue fast staining method. The further in vitro experiment results proposed that the adsorbed proteins affected the interactions between silanes-MNPs and cancer cells, suggesting that the effects of proteins from culture medium on surface property of MNPs should be taken into account in relatively biological investigations.
1-octadecene was purchased from Alfa Aesar. 3-Aminopropyltriethoxysilane (NH2-silane),n-(trimethoxysilylpropyl)ethylene diamine triacetic acid (45% in water)(COOH-silane), and 2-[methoxy-(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane) were obtained from ABCR gmbh. Coomassie blue (G-250) was purchased from Amerosco. The other chemicals were analytical reagents and purchased from Shanghai Chemical Reagent Corporation, China. All chemicals were used as received.
Synthesis of Monodisperse Fe3O4MNPs
The synthesis of monodisperse Fe3O4 MNPs was based on a previously reported study . In brief, 1.08 g (4 mmol) of FeCl3 · 6H2O and 3.65 g (12 mmol) of sodium-oleate were dissolved in a mixture solvent containing 6 mL water, 8 mL ethanol, and 14 mL hexane. The resulting solution was stirred for 4 h at 70 °C. Then, the upper organic layer containing iron-oleate was washed three times with 3 mL water in a separatory funnel. After the water and hexane were evaporated off under vacuum, solid iron–oleate was collected. The obtained 2.8 g (3 mmol) iron–oleate and 0.42 g (1.5 mmol) of oleic acid thus obtained were dissolved in 15 g (50 mmol) 1-octadecene. The mixture was heated to 320 °C at a constant heating rate of 3.3 °C/min and maintained at that temperature for 30 min. Later, the resulting solution was cooled, precipitated by addition of excess ethanol. Then, the precipitate containing MNPs was washed 4–5 times with a mixture of hexane and acetone. Finally, MNPs were dispersed in hexane to obtain a black solution.
Surface Functionalization of MNPs with Silanes
In a glass container under ambient conditions, surface silanes functionalization of MNPs was performed according to the reported study with minor modification . In a typical experiment, 0.5% (v/v) silane solution was added to a dispersion of MNPs in hexane (30 mg in 10 mL) containing 0.01% (v/v) acetic acid. The mixture was shaken for one day and eventually a dark brown precipitate was obtained. During the magnetic separation step, MNPs were washed three times with hexane to remove excess silanes, followed by washing with ethanol to remove hexane. Then the product (silane-MNPs) was washed three times with deionized water and finally dispersed in water. Iron quantitative assessment of the yield of silane-MNPs was performed using inductively coupled plasma emission spectroscopy (ICP) .
Silanes-MNPs Treated for Zeta, UV–Vis Absorbance Measurements, and Cell Culture
To obtain protein-silane-MNPs conjugates, 1 mg of silane-MNPs (determined as iron) was incubated in 10 mL RPMI-1640 medium plus 10% FCS for 12 h. Then, the product thus obtained was separated by a magnet and washed three times to eliminate residual serum. The remnant RPMI-1640 medium was used for Coomassie blue fast staining and UV–vis measurements.
Cell Culture and Quantitative Cellular Uptake of MNPs
Human hepatoma cells (SMMC-7721) were used in in vitro experiments. Cells were cultured at 37 °C in a 5% CO2atmosphere, in a 24-well culture plate containing 0.6 mL RPMI-1640 medium plus 10% FCS. After washing twice with PBS to eliminate fetal calf serum, the cells were incubated with silane-MNPs and protein-silane-MNPs in RPMI-1640 without FCS for 12 h, respectively. Iron quantitative cellular uptake of nanoparticles was also determined based on ICP method. Three replicates were measured and the results were averaged with standard deviation.
The size and morphology of the particles were determined by transmission electronic microscopy (TEM, JEOL, JEM-200EX) operating at 120 kV. Samples were dropped from hexane onto a carbon-coated copper grid and dried under room temperature. IR spectra were recorded on a Nicolet Nexus 870 FT-IR spectrometer and powder samples were dried at 100 °C under vacuum for 24 h prior to fabrication of the KBr pellet. Spectra were recorded with a resolution of 2 cm−1. Surface charge measurements were performed on a Zeta Potential Analyzer (Delsa 440SX, Beckman Coulter). ICP results were collected on a microplate reader (Model 680, Bio-RAD).
UV–vis spectra were recorded using a U-4100 spectrophotometer. To prepare Coomassie blue solution, 100 mg G-250 was dissolved in a mixture containing 45 mL ethanol and 5 mL water, followed by addition of 100 mL 85% phosphoric acid. The resulting solution was diluted to 1,000 mL with water. For UV–vis absorbance spectra measurements, 0.5 mL RPMI-1640 medium plus 10% FCS and 0.5 mL remnant RPMI-1640 medium which has been treated by silane-MNPs were added into 5 mL Coomassie blue solution, respectively. The resulting solution turned from black to blue and showed absorbance peak at 595 nm.
Results and Discussion
Synthesis, Surface Functionalization, and Characterization of MNPs
Adsorption of Proteins from Culture Medium and its Effects on Surface Property of Silanes-MNPs
In summary, we investigated the effects of proteins from culture medium on the surface properties of MNPs functionalized with silanes bearing various functional groups such as NH2, PEG, and COOH. Despite the distinct surface properties, all the silanes-MNPs exhibited negative surface charges at neutral pH and approximate isoelectric points (IEP) when incubating in cell culture medium. This is due to adsorption of proteins onto the surface of silanes-MNPs and formation of protein-silanes-MNPs conjugates. Since surface was in direct contact with cells, surface property of MNPs is very important in the investigations on the interactions between MNPs and cancer cells. Therefore, we proposed that adsorbed proteins could play a great role in cellular uptake of MNPs by cancer cells, which was confirmed by in vitro cell experiments. The investigation on the effects of proteins from culture medium on MNPs’ surface property revealed in this study might be helpful both in in vitro and in vivo experiments.
This study has been carried out under financial support of the National Natural Science Foundation of China (No. 60571031, 60501009 and 90406023) and National Basic Research Program of China (No. 2006CB933200 and 2006CB705600). Open Project Foundation of Laboratory of Solid State Microstructures of Nanjing University is greatly appreciated.