Preferential adsorption of fetal bovine serum on bare and aromatic thiol-functionalized gold surfaces in cell culture media
Graphical abstract
Highlights
► Adsorption of serum was examined on bare and aromatic thiol-functionalized gold surfaces. ► Quartz crystal microbalance data indicated that fetal bovine serum is the dominant adsorbate species on gold surfaces. ► Cellular uptakes of aromatic thiol-functionalized gold particles were checked by surface-enhanced Raman scattering.
Introduction
Engineered nanoparticles (NPs) are used in a wide variety of applications, including targeted drug/gene delivery, cellular imaging, and biomedical sensors and therapies [1], [2]. However, there has been a growing recognition of the potential risks and adverse effects of NPs on living systems [3], [4], [5]. The surfaces of NPs that are in contact with a biological environment are in dynamic exchange with biomolecules such as proteins and lipids [6], [7]. Much attention has been paid to understanding the interactions of inorganic NPs and biological systems, including protein corona, in nanotherapeutics [8]. Protein–NP interactions are dissimilarly mediated by the different composition of cellular media. When seeking to understand controlled delivery into a cell, one must first understand the interactions of proteins and NPs.
Gold nanoparticles (AuNPs) have attracted much attention in the past several decades due to their stability and optical properties [9], [10], [11], [12], [13], [14]. Recent reports on the biological applications of AuNPs have focused on the effects of biocompatibility, uptake, and sub-cellular distribution of AuNPs [15], [16]. In order to obtain the three-dimensional localization information on the uptake of NPs into cells, confocal microscopy with a sufficient resolution for the z-stack direction is required [17], [18], [19]. Dark-field microscopy (DFM) is an effective imaging tool for noble metal nanostructures [20]. Since its discovery, surface-enhanced Raman scattering (SERS), an ultra-sensitive spectroscopic tool for interface studies, has been widely used for biological sensing or molecular imaging near the surface of nanostructure assemblies [21], [22], [23]. The SERS method offers several advantages due to the different Raman signatures in the high throughput screening of various biomolecules with narrow bandwidths, thus avoiding spectral overlaps. This work was motivated by the lack of information on NP–protein interaction and their uptake behaviors inside cells using spectroscopic tools. In this work we performed a confocal SERS and DFM study of Au NP embedded A549 human lung carcinoma cells to better monitor the inside of mammalian cells with a micrometer z-depth resolution. In order to monitor the intracellular AuNPs using Raman spectroscopy, aromatic thiols bearing NH2, OH, and COOH are self-assembled onto AuNPs, whose localization is roughly checked by DFM. We counted the number of AuNPs inside a single cell and used z-stack SERS spectra to estimate their uptake tendency. Our results suggest that the aromatic thiol-modified AuNPs enter the cell after coating the constituents in the serum.
Section snippets
Preparation of aromatic thiol-modified AuNPs
The colloidal dispersions of negatively charged AuNPs were prepared by the citrate reduction method [24]. First, 30 mL of aqueous 14 mM HAuCl4 (99.9%, Sigma Aldrich) solution was boiled for the Au(−) NP sample. Approximately 3 mL of 1% sodium citrate solution was then added to the HAuCl4 solution under vigorous stirring, and boiling was continued for ca. 1 h. 4-carboxylic benzene thiol (CBT, 4-mercaptobenzoic acid, 90%) and 1,4-phenylenediisocyanide (PDIC, 99%) were purchased from Sigma Aldrich
Self-assembly of serum on Au surfaces
AuNPs are presumed to adsorb the proteins or other constituents of the serum in the media. DFM combined with micro-Raman spectroscopic tools are employed to monitor the uptake of serum-coated AuNPs. DFM is first used to localize AuNPs inside a single cell. Aromatic thiols are self-assembled to obtain the z-stack SERS spectra from AuNPs. Fig 2 shows TEM and AFM images.
Since there are many constituents in the serum and medium, we chose to use the bovine serum albumin (BSA) protein as an example
Summary
This QCM study indicates that fetal bovine serum is the dominant constituents in a cell growth RPMI medium. We examined the cellular uptake of aromatic thiol-derivatized AuNPs in a single mammalian A549 cell using SERS combined with DFM. By identifying the location of the AuNPs with DFM, an intracellular localization assay was possible using different Raman dyes with unique vibrational marker bands. It was found that these AuNPs could enter the cell without much difference depending on the
Acknowledgments
We acknowledge the financial support from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology Technology (2011-0001316, 2011-0003159, 2010-0026100) and the Development of Characterization Techniques for Nano-materials Safety Project of KRCF.
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