The nanoparticle identity card: on the characterisation of the protein corona by scattering and microscopy techniques
The biological behaviour of nanoparticles within our body is partly determined by a particle-specific protein layer that rapidly forms in contact with physiological media. Reliable methods to understand this layer or "protein corona" need to be developed if nanoparticles are to be implemented in applications. Ultimately, in situ characterisation of the protein corona could improve lives by allowing to implement and design nanomaterial-based medical therapies. However, the large number and variability of proteins, their low imaging and scattering contrast, combined with the rapid formation kinetics of the protein corona make reliable and reproducible studies a complex task.
The studies conducted in this thesis investigate the protein corona of particles that differ: in composition (gold and silica); size (50 to 2000 nm); shape (spherical, faceted and rod-shaped) and structural length scale (atomic and mesoscale). One aim of this thesis is to visualise the reversible and rapidly exchanging soft corona of weakly bound proteins and the inner, irreversible hard corona of strongly bound proteins around the different nanoparticles. Advanced imaging is performed using various electron microscopy techniques such as scanning electron microscopy, transmission electron microscopy and cryo-transmission electron microscopy. They demonstrate that the dimensions and morphology of the PC are highly dependent on the size and the shape of the NPs, showing an extended soft corona for faceted and high aspect ratio mesoporous silica particles.
Additionally, another goal is the use of scattering techniques such as small angle X-ray scattering, surface enhanced Raman scattering and dynamic light scattering, in combination with mass spectrometry to reveal the composition of the different protein coronas and its effects on their biological endpoints (e.g. cell uptake, potential cytotoxicity). Using these ex-situ techniques showed that the manipulation of the PC with single proteins such as fibrinogen can alter the cellular uptake of gold nanoparticles as well as their ability to generate intracellular reactive oxygen species. This is significantly different from the results that pure gold nanoparticles show in cellular uptake studies, which generate minimal reactive oxygen species once internalised by cells.
Moreover, another goal of this thesis is to study the in situ formation of the protein corona around different nanoparticles. Therfore, in situ small angle X-ray scattering was used to measure the dynamics of the protein corona formation. Sequential protein addition indicated that the protein corona influences the size and shape of gold nanoparticles as well as mesoporous silica particles within 10 seconds of contact with the biological media, due to agglomeration caused by protein-particle and protein-protein interactions. These behaviours depend on the order and incubation time that proteins are exposed to the nanoparticle surface.
Evading the immune system is an important goal in drug delivery and theranostics. Controlling the PC could allow the design of a protein corona that is mediating increased cell uptake and targeting. Overall, this thesis gives an insight on how the specific manipulation of the protein corona could be utilised as a drug delivery platform, leaning away from the common idea of seeing it as an obstacle in nanomedicine. New questions that arise from the completion of this thesis are:
1. What are the mechanisms govern the specific adsorption of proteins to facets in mesoporous silica particles?
2. How can these mechanisms be exploited to predict and design the immunological behaviour of nanoparticles?
3. How can electron tomography be improved in terms of contrast and resolution in cryotransmission electron microscopy to reveal folding and structural changes in the protein corona?
4. Do the lessons learned about the in vitro and ex-situ characterisation techniques lead to a better in vivo characterisation of the protein corona?