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Synthesis and self-assembly of Ferritin-based novel Biohybrid Nanoparticles

Final Activity Report Summary - SYSAFEBION (Synthesis and Self-Assembly of Ferritin-Based Novel Biohybrid Nanoparticles)

Viruses have always been enemies of mankind, but recent advances in chemistry related to biological structures are starting to change this perspective. Viral capsids and other protein cages, such as (apo-)ferritin, offer robust, monodisperse and highly organised supramolecular structures to be utilised as nano-scaffolds. Moreover, the protein cages that are available in nature show a broad spectrum of shapes and sizes, ranging from 12 to 500 nm for icosahedral capsids and to a length of more than 2 µm in the case of tubular viruses.

For these reasons, protein cages could be used to carry out chemical processes at three different levels:

1. in the interior of their inner cavity
2. on their exterior surface, and
3. at the interface where their constituent protein subunits interact to self-assemble.

The potential advantages of this research were many. Synthesising inorganic and organic materials inside these hollow architectures allowed gaining control over the structure, size and morphology of these materials. The external surface of protein cages that contained some kind of material could be functionalised with organic polymers, providing solubility in organic solvents and better processing properties. Finally, it was also interesting to study the self-assembly of viral proteins in the presence of synthetic polymers, which could be encapsulated, giving raise to biohybrid virus-like particles (VLPs) of different sizes and shapes.

The protein cage that we chose was the cowpea chlorotic mottle virus (CCMV) capsid, which was formed by 180 protein subunits of 20 kDa each. This virus showed the remarkable ability to disassemble and assemble depending on the pH conditions. CCMV virions disassembled into protein dimers and ribonucleic acid (RNA) when the pH of the media was raised above 7.5. After RNA removal, the purified viral coat protein (CP) subunits could self-organise and form empty capsids in case the pH was decreased to 5. This pH-dependent behaviour was used by us and others to encapsulate different species such as proteins, inorganic nanoparticles and anionic polymers.

During the course of this Marie Curie project, the use of the CCMV capsid as a nanoreactor was explored. The well-known magnet Prussian blue has been synthesised in the interior cavity of CCMV, as a method for the preparation of monodisperse magnetic nanoparticles. The organisation of these particles into a hexagonal pattern on surfaces was also achieved, suggesting their potential use in information storage devices. Furthermore, the exterior of the CCMV-Prussian blue particles was modified with decylamine chains and Traut´s reagent, i.e. a thiol-containing reactant. In the first case, the particles became soluble in organic solvents. In the latter one, further functionalisation with gold nanoparticles was possible, revealing an electronic interaction between both types of materials which was under study by the time of the project completion. All these experiments demonstrated the very rich and versatile chemistry that could be developed using the CCMV capsid.

In the same line of thought, we tried to use CCMV as a nanoreactor to control the structural features, such as molecular weight and polydispersity, of polymers synthesised inside the viral cavity. The photo-polymerisation of styrene sulfonate, with eosine Y as the photo-initiator, was selected as the model polymerisation system. Yet a great surprise was observed. We had important indications, from gel permeation chromatography (GPC) and matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) spectrometry, to believe that the special chemical environment that was present inside CCMV had favoured a very rare side reaction by which polyvinyl sulfonate was formed instead of polystyrene sulfonate (PSS). Further experiments were carried out to provide stronger evidence in this respect.

It was shown, from the experiments described above, that the very precise structure of CCMV could be used to determine the structural features of materials prepared inside its capsid. The opposite approach was also possible, i.e. to employ synthetic templates to control the types of assemblies that the viral protein could produce. This was of importance for the synthesis of functional biohybrids.