RESUMEN
Nanoparticles in contact with proteins form a "corona" of proteins adsorbed on the nanoparticle surface. Subsequent biological responses are then mediated by the adsorbed proteins rather than the bare nanoparticles. The use of nanoparticles as nanomedicines and biosensors would be greatly improved if researchers were able to predict which specific proteins will adsorb on a nanoparticle surface. We use a recently developed automated workflow with a liquid handling robot and low-cost proteomics to determine the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200 nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. In addition to these findings, we describe two methodological advances to help reduce the cost associated with protein corona experiments. We have automated the digest step necessary for proteomics and measured the variability between triplicate samples at each stage of the proteomics experiments. Overall, these results demonstrate the importance of understanding the multiple parameters that influence corona formation, provide new tools for corona characterization, and advance bioanalytical research in nanomaterials.
Asunto(s)
Nanopartículas , Corona de Proteínas , Nanomedicina , Proteómica , Albúmina Sérica BovinaRESUMEN
Nanoparticles used in biological settings are exposed to proteins that adsorb on the surface forming a protein corona. These adsorbed proteins dictate the subsequent cellular response. A major challenge has been predicting what proteins will adsorb on a given nanoparticle surface. Instead, each new nanoparticle and nanoparticle modification must be tested experimentally to determine what proteins adsorb on the surface. We propose that any future predictive ability will depend on large datasets of protein-nanoparticle interactions. As a first step towards this goal, we have developed an automated workflow using a liquid handling robot to form and isolate protein coronas. As this workflow depends on magnetic separation steps, we test the ability to embed magnetic nanoparticles within a protein nanoparticle. These experiments demonstrate that magnetic separation could be used for any type of nanoparticle in which a magnetic core can be embedded. Higher-throughput corona characterization will also require lower-cost approaches to proteomics. We report a comparison of fast, low-cost, and standard, slower, higher-cost liquid chromatography coupled with mass spectrometry to identify the protein corona. These methods will provide a step forward in the acquisition of the large datasets necessary to predict nanoparticle-protein interactions.
Asunto(s)
Nanopartículas/química , Corona de Proteínas/análisis , Proteómica/métodos , Animales , Macrodatos/economía , Bovinos , Humanos , Nanopartículas/ultraestructura , Ovalbúmina/análisis , Proteómica/economíaRESUMEN
Inhalation is a major exposure route to nanoparticles. Following inhalation, nanoparticles first interact with the lung lining fluid, a complex mixture of proteins, lipids, and mucins. We measure the concentration and composition of lung fluid proteins adsorbed on the surface of titanium dioxide (TiO2) nanoparticles. Using proteomics, we find that lung fluid results in a unique protein corona on the surface of the TiO2 nanoparticles. We then measure the expression of three cytokines (interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2)) associated with lung inflammation. We find that the corona formed from lung fluid leads to elevated expression of these cytokines in comparison to bare TiO2 nanoparticles or coronas formed from serum or albumin. These experiments show that understanding the concentration and composition of the protein corona is essential for understanding the pulmonary response associated with human exposure to nanoparticles.