Nanoparticles hold great promise for many biological applications, most notably in the delivery of therapeutic drugs. Proteins play a central role in biological systems, but as yet there has been no simple method of working out the structure and shape of nanoparticle-protein complexes. This has proven to be a major limiting factor in the development of biological applications for nanoparticles.
Now a study published in Nanoscale, using the Circular Dichroism beamline (B23) , has taken a major step towards solving this problem by discovering that the structure of the human serum albumin protein changes when it becomes part of this nanoparticle-protein complex (Fig. 1). Dr Luigi Calzolai from the Joint Research Centre at the European Commission led this study, which was also able to measure the affinity of a protein for a gold nanoparticle.
A better understanding of these nanoparticle-protein complexes is essential to pave the way towards nanoparticle drug delivery, and as part of their collaboration with the newly established European Nanomedicine Characterization Laboratory (EU-NCL), the research team hope to use the methods developed in this study to characterise nanomedicine drug-delivery systems.
When nanoparticles are introduced into a biological system (e.g. into the bloodstream), they can be quickly surrounded by proteins of many different kinds, as the immune system recognises a foreign entrant in the body. Understanding these protein-nanoparticle interactions is important both for applications where this immune system recognition might need to be bypassed (e.g. when the immune system might work to destroy the nanoparticle and the useful cargo that it is carrying), and for applications where the nanoparticle needs to chemically bind with biological proteins in order to exert the desired effect.
But until now, it has been difficult to quantify even basic measures such as the number of protein molecules bound to each nanoparticle, and how stable a particular nanoparticle-protein complex is. The function of proteins is critically dependent on their structure, so it is also important to find out whether and how binding to nanoparticles changes the arrangement of molecules that make up a protein.
Fig. 1: The structure of Human serum albumin (HSA) complexed with 6 palmitic acid molecules (from PDB 1E7H).
A team from the Joint Research Centre at the European Commission and the University of Lyon led by Dr Luigi Calzolai at the Joint Research Centre used the intense light produced by Diamond Light Source to tackle these problems.
“We have previously used the B23 beamline to look at the same problem,” said Dr Calzolai. “But we have refined our methods and have been able to separately measure the properties of protein molecules bound to a gold nanoparticle versus the free, unbound protein.”
The research team first measured the size and density of the nanoparticleprotein complex. From these measurements, they worked out the number of proteins bound to each nanoparticle.
They then used measurements of circular dichroism spectra on the B23 beamline to work out the secondary structure and folding properties of the protein molecule in this nanoparticle-protein complex. This technique takes advantage of the fact that when chiral molecules (such as proteins) interact with circularly polarised light (CPL), they absorb the right and left handed CPL to different extents. Different protein structural elements therefore have different and characteristic circular dichroism spectra.
“Our experiments really pushed this technique to the limit, and we are very thankful to Professor Giuliano Siligardi and Dr Rohanah Hussain from B23: their expertise with circular dichroism was very useful, and we have a very productive long-term collaboration with them,” said Dr Calzolai.
The research team also incubated nanoparticles with proteins at different ratios, and then measured the amount of protein bound to each nanoparticle (Fig. 2). By using this method, the group also demonstrated a technique for working out the binding affinity between specific proteins and nanoparticles.
Fig. 2: Model of the AuNP–HSA complexes. At a low protein–NP ratio (left) the bound HSA molecules occupy less than 50% of the available binding sites and their secondary structure is more distorted. At a high protein-NP ratio (right) complete coverage of the nanoparticle surface is reached. Capomaccio et al. – Published by The Royal Society of Chemistry.
Only a tiny fraction of proteins bind to nanoparticles, so any measurement technique needs to be sensitive enough to pick up signals from this small sample. “On a standard instrument, we use a sample cell which is only about 1 cm long,” said Dr Calzolai. “But at Diamond, the light intensity is so much stronger, the beam is smaller and highly collimated that we can use a 10 cm long sample cell with 3 mm aperture increasing our sensitivity by a factor of 10 and decrease by 30 times the sample volume.” The extremely high photon flux also means that the gold nanoparticles used in this experiment are much more visible, despite their light absorption properties, which make their detection difficult using bench-top CD instruments.
The research team now hope to use the methods they describe in this paper to characterise other potential nanomedicine drug-delivery systems. Many such systems include a protein component – e.g. a single chain antibody or peptide to target the drug delivery specifically to cancer cells.
“We now want to find out how the structure of the nanoparticle-bound antibody changes - if it folds or unfolds in a different way, for example.”
Capomaccio et al. Determination of the structure and morphology of gold nanoparticle-HAS protein complexes. Nanoscale (2015). DOI:10.1039/C5NR05147A
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