Date Published: June 14, 2019
Publisher: Public Library of Science
Author(s): Stefán B. Gunnarsson, Katja Bernfur, Ulrica Englund-Johansson, Fredrik Johansson, Tommy Cedervall, Amitava Mukherjee.
New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates’ morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.
The unique properties of nanostructured materials have led to an ever increasing number of applications in various fields, e.g. electronics, cosmetics, heavy industry, and in pharmaceuticals [1–3]. Nanomaterials are all materials that have at least one dimension in the range of 1–100 nm which gives them properties that are between atomic and bulk. One of the main benefits of nanomaterials is their high surface to volume ratio compared to bulk material, i.e. less material is needed for the same surface activity. Since nanomaterials are defined by their size, they can differ in chemical composition, shape, and electronic and optical properties. Utilization of the enormous potential of nanomaterials calls for unique and highly specific approaches in ensuring their safe application. In biological context, an important property of nanostructures is that their size is the same as many biomolecules, e.g. proteins . While some designed nanomaterials are produced with the aim of in vivo use, e.g. in diagnostics and therapeutics, other nanomaterials are intended for applications outside of the human body, e.g. in heavy industry and electronics. For both biological and non-biological designed nanomaterials, it is important to understand the interactions and effect of the material on organisms in the case of both intended and unintended exposure.
To monitor the interaction of nanoparticles with components of cell culture media (CCM), we incubated gold nanoparticles, with a diameter of 20 nm, in protein rich (RPMI) and protein poor (expansion) media at an Au NP concentration of 2.8 μg/mL. RPMI is supplemented with 10% fetal bovine serum (FBS) (approximately 7 mg/ml proteins) while protein poor CCM only contains proteins in the form of growth factors at concentrations in the ng/mL range. The protein concentration difference in the two media is therefore six orders of magnitude. However, the amino acid, carbohydrate, and salt concentrations are high in both CCM. At low NP concentrations and relatively high concentration of various biomolecules like proteins, carbohydrates, lipids, amino acids, etc. the NP surface will quickly be covered with biomolecules. It is important to consider that NP aggregation that is dependent on direct contact between the surfaces of two Au NPs will happen slower at lower concentration. How long it takes for the system to reach equilibrium will ultimately determine what cells are exposed to and the NPs toxicological impact. Information about the aggregation state and aggregate morphology is crucial when assessing how fast the nanomaterial reaches the cell, if different fractions of the nanomaterial will reach the cell by sedimentation or diffusion, and available surface on the nanostructure for contact with the cell. For example, spherical nanoparticles are more readily taken up by cells than rod-like particles . Upon interaction with biomolecules from the environment, citrate is displaced on the Au NP surface, which may trigger a chain like fractal aggregation . However, a chain of spherical Au NPs would not necessarily behave like the nanorods. The more information we can gather on the system’s behavior, the better we can explain the cell’s response. For a detailed description of the NP behavior, we combine analytical detection methods of different qualities and apply them in a synergistic way.
In protein poor CCM, a slow development of fractal aggregation was observed. The first indication of the aggregate morphology was observed when comparing the sedimentation and diffusion behavior of the aggregates by DCS and DLS (Figs 1a and 2a), respectively. Slow sedimentation of the aggregates indicated non-spherical morphology. The appearance of two peaks in the absorbance spectra, around 540 and 650 nm supports our conclusion and cryo-TEM pictures confirm the aggregate morphology. Finally, ATR-FTIR confirms the presence of biomolecules known to induce fractal aggregation on the NP surface. In protein rich CCM, the NPs were stable towards aggregation. The high relative concentration of proteins compared to the NPs made it impossible to measure their actual hydrodynamic diameter by DLS as we could only detect a shift to larger species in the average size distribution. Combining DLS and DCS, it is possible to rule out aggregation as the cause of this increase in size, since the smaller apparent diameter is caused by the adsorption of relatively low density biomolecules to the high density Au NPs. The UV-Vis absorbance spectrum supports this conclusion since there is only one stable peak observed at 534 nm. Characterizing the complexes formed when Au NPs in a very low concentration interact with biomolecules is a key step in assessing their biological impact. In order to study the development of the biomolecular corona in concentrations similar to those used in cell studies, we needed to account for the low concentration with a combination of analytical methods. Doing so we reveal information that no single method can.