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Protein corona formation on polymer-coated nanoparticles

Scientific organization
Institute of Biology and Biomedicine Lobachevsky State University of Nizhni Novgorod
Academic degree
Junior researcher
Scientific discipline
Life Sciences & Medicine
Protein corona formation on polymer-coated nanoparticles
Biofuncational nanomaterials are ushered into the era of theranostics offering new possibilities for diagnosis and treatment. Their surface grafted with functional biomolecules is swiftly remodelled as soon as they enter biological fluids, such as blood serum – the process termed “protein corona”. We report on investigation of how the protein corona is formed on polymer-coated upconversion nanoparticles (UCNP) by using transmission electron microscopy, dynamic light scattering, protein assaying and fluorescence correlation spectroscopy.
upconversion nanoparticles, protein corona, theranostics

A. B. Kostyuk1, L. Liang2, R. Zhang2, A. A. Tretyakov1, A. V. Zvyagin1,2

1 Laboratory of Optical Theranostics, Lobachevsky State University of Nizhni Novgorod, Nizhny Novgorod, Russia

2 ARC Centre of Excellence “BioPhotonics at Nanoscale”, Macquarie University, Sydney, Australia

E-mail: andrei.zvyagin@mq.edu.au

   Development of new approaches for the diagnosis and therapy of tumours (taken together, termed theranostics) - one of the most dynamic areas of biomedicine, where new nanomaterials afford new opportunities. The nanomaterial merits include: programmability of their physical and chemical properties; abundance of reactive functional groups on the surface; large effective surface area; optimum size, which determines preferential accumulation of nanoparticles (NPs) in tumour tissue 1. Understanding of how engineered nanomaterials interacting with biological systems and environment is presently a most exciting research topic at the interface between nanotechnology and the life sciences. 2-4 It is increasingly being accepted that NPs encountering biological medium are swiftly coated by a biomolecular adsorption layer, the so-called “protein corona”. 5, 6 Consequently, the molecular machinery of a living cell or organism will – at least initially – interact with the corona rather than the bare NP, making it a key determinant of the biological response of NP exposure. Indeed, studies have reported that protein adsorption onto NPs affects their cellular uptake efficiency, controls the internalisation mechanism, 7 and modulates pathobiological effects. 8 Therefore, a profound knowledge of the protein corona, including its composition, structure, dynamics and thermodynamics, is of fundamental importance for the safe and well controlled application of NPs.

   As a model NP, we choose upconversion nanoparticles (UCNPs) allowing background-free optical imaging 9, which make them attractive for applications in life sciences 10. UCNPs in the form of hexagonal crystallites NaYF4 doped with Yb3+ and Er3+ or Tm3+ were synthesised in the size range of 20 – 30 nm by a modified solvothermal method 11 [see Figure 1 (left panel)]. UCNPs were coated with three types of polymers: positively charged polyethylenimine (PEI), negatively-charged polyacrylic acid (PAA) and neutral-negative polyethylene glycol (PEG), which is regarded as excellent dispersant. Although incubation with mono-component plasma protein solution (e.g. bovine serum albumin - BSA) displayed well-studied formation of a monolayer of BSA on the surface of UCNP [Figure 1 (middle panel)], its incubation in fetal bovine serum (FBS) displayed profound protein corona formation, followed by some degree of aggregation [Figure 1 (right panel)].

Figure 1. TEM images of UCNPs-PAA, shown in left panel. Middle panel, TEM image of UCNP-PAA incubation with BSA, with an arrow showing a monolayer of BSA. Right panel, TEM image of UCNP-PAA incubated in cell culture medium at 37 °C for 24 h stained with 2% phosphotungstic acid (PTA).

   Our protein assaying study corroborated the TEM observation, as shown in Figure 2. It shows profound effect of the UCNP-polymer surface charge. In particular, UCNP-PEI acquired almost ten-fold of protein content as compared to that of UCNP-PEG and –PAA. Figure 2 (inset) also shows that the protein binding occured within minutes, which is in agrement with the literature 12.

Figure 2. Temporal development of protein corona on UCNP-PEI, UCNP-PAA, and UCNP-PEG at 10 min, 30 min, 1 h, 2 h, 4 h and 24 h, measured by Micro BCA assay. Protein corona was formed by incubating 1 mg of UCNP in 1 mL of cell culture medium (DMEM+10% FBS) at 37 °C for different time points. After removal of soft corona via three wash steps, the hard protein corona mass was estimated by colometric BCA assay using the BSA standard curve. Abbreviations: BCA, bicinchoninic acid.

   The zeta-potential measurement showed the surface charge became more negative. The protein corona of FBS origin enhanced the surface binding and internalisation of all types of UCNPs.

   We will report on our results on the protein corona formation by means of fluorescence correlation spectroscopy, which provides a very valuable in situ measurements means.

   This work was supported by grant No. 14.Z50.31.0022.




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