(497b) Elucidating Protein Corona Formation on Nanoparticles in Complex Biological Fluids

Engineered nanoparticles have increased in prominence for biological sensing [1], imaging [2], and gene delivery [3] applications owing to their distinctive optical and physical properties. The critical – and often overlooked – challenge with these nanoscale tools is understanding the fundamental mechanisms of interaction between the nanoprobes and the system they are designed to query. When a nanoparticle enters a biological system, the surface becomes coated with proteins to form the ‘protein corona’. Binding of proteins to the nanoparticle not only affects the structure and function of the proteins, but has the added consequence of unpredictably masking and re-defining the nanoparticle identity, potentially preventing the nanoparticle from carrying out its designated function.

To apply such probes in biological systems, it is critical to understand: (i) what coats a nanoparticle upon exposure to a biological system, (ii) how this binding process occurs, and (iii) why these particular biomolecules coat the nanoparticle. Existing studies correlating nanoparticle properties to corona formation concentrate almost exclusively on systems of spherical model nanoparticles in blood plasma and solely answer the question of ‘what’ is coating the nanoparticles without rigorously delving into the underlying mechanistic ‘how’ and ‘why’ [4], [5]. The work presented herein develops and tests a novel view of protein corona formation around nanoparticles for applications in blood plasma and cerebrospinal fluid. The method involves (i) experimentally investigating protein corona composition, (ii) experimentally probing the thermodynamics and kinetics of protein binding, and (iii) interpreting experimental results with the framework provided by colloid and surface science theory.

We present results on protein corona composition as determined by 2D SDS PAGE and liquid chromatography-mass spectrometry. The assay is translatable from ubiquitously-studied polystyrene nanoparticles (PNPs) to electrostatically-functionalized single-walled carbon nanotubes (SWCNTs), and protein binding is determined for both PNPs and SWCNTs in two biological fluids of interest: blood plasma and cerebrospinal fluid. Key proteins found to be conserved in the corona across both biofluids include fibrinogen and certain apolipoproteins, which play roles in blood clotting and lipid transport, respectively. Within this selective adsorption assay, incubation conditions including temperature, ionic strength, and dynamics are varied to provide insights on the factors governing protein adsorption and corona formation. Notably, the incubation solution ionic strength plays a critical role in proteins adsorbing to the polymer-encapsulated SWCNTs but has little effect for the case of PNPs. Based on our experimental results, we posit the dominant colloidal interactions to govern corona formation to be (i) hydrophobic interactions between the SWCNT surface and hydrophobic protein domains, (ii) pi-pi stacking between the graphitic domains on the SWCNT surface and aromatic amino acids on the protein, and (iii) electrostatic interactions between the charged polymer encapsulating the SWCNT and the charged protein and salts in solution. Further kinetic and thermodynamic quantification of the time-dependent protein corona composition, and timescales upon which the protein corona forms, will establish parameters for the predictive design of nanoparticle corona formation. This knowledge will inform figures of merit for the design and synthesis of nanotechnology-based tools including nanosensors [6] and nanodrugs [7] to be applied in protein-rich environments, enabling corona formation to be taken into account ab initio.

References

[1] J. Zhang et al., “Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes,” Nature Nanotechnology, vol. 8, no. 12, pp. 959–968, Nov. 2013.

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[3] G. S. Demirer et al., “High Aspect Ratio Nanomaterials Enable Biomolecule Delivery and Transgene Expression or Silencing in Mature Plants,” bioRxiv, p. 179549, Jan. 2018.

[4] M. P. Monopoli, C. Åberg, A. Salvati, and K. A. Dawson, “Biomolecular coronas provide the biological identity of nanosized materials,” Nature Nanotechnology, vol. 7, no. 12, pp. 779–786, Dec. 2012.

[5] M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, and K. A. Dawson, “Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts,” Proceedings of the National Academy of Sciences, vol. 105, no. 38, pp. 14265–14270, 2008.

[6] A. G. Beyene, I. R. McFarlane, R. L. Pinals, and M. P. Landry, “Stochastic Simulation of Dopamine Neuromodulation for Implementation of Fluorescent Neurochemical Probes in the Striatal Extracellular Space,” ACS Chem. Neurosci., vol. 8, no. 10, pp. 2275–2289, Oct. 2017.

[7] J. Shi, A. R. Votruba, O. C. Farokhzad, and R. Langer, “Nanotechnology in Drug Delivery and Tissue Engineering: From Discovery to Applications,” Nano Lett., vol. 10, no. 9, pp. 3223–3230, Sep. 2010.