Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

David Paramelle,David G Fernig,Paul Free,Sierin Lim,Tao Peng,Nikodem Tomczak

doi:10.1371/journal.pone.0162848

Abstract

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles.

Highlights

Protein cages and virus-like particles are highly structured and stable homo- and hetero-supramolecular protein self-assemblies.[1]
We evaluate the specific internalisation via affinity binding of single small gold nanoparticles into porous protein cages
To impart the affinity for the oligohistidine sequences present inside the E2 protein cage’s core, we passivated the surface of small gold nanoparticles with a self-assembled monolayer (SAM) coating functionalised with NiNTA moieties (Fig 2B)

Summary

Introduction

Protein cages and virus-like particles are highly structured and stable homo- and hetero-supramolecular protein self-assemblies.[1]. During the last two decades, materials scientists have intensively investigated the manipulation of protein cages for the encapsulation of single inorganic nanoparticles into their core to design novel hybrid bioinspired nanoparticles.[9,10] As such, the preparation of hybrid nanoparticles has been validated for a variety of inorganic cores, e.g., noble metals[11,12] or metal oxides,[13] and with different types of protein cages and virus-like particles, e.g., cowpea chlorotic mottle virus[14] or ferritins.[15,16,17,18,19,20] Interestingly, the development of protein cage-based hybrid nanoparticles[21] allowed one to combine into the hybrid material the physicochemical properties of the inorganic nanoparticles, e.g., plasmon resonance, fluorescence, with properties of biomaterials, e.g., biocompatibility Such hybrid nanoparticles represent an opportunity for advanced nanotechnology applications in the fields of molecular imaging, [13,16,22] nano-electronics[23] and catalysis.[24,25]

Methods

Results

Conclusion