It has not quite got the cultural cachet of a nuclear apocalypse, but everyone knows how cyberwarfare goes. A spy with a thumb drive; an unprotected USB; secrets stolen, virus shared: civilisation in ruins. But wait. Stop catastrophising. Let’s start again and see if it does not turn out differently. This is a spy story, remember. It’s got to have a twist.

The one with the thumb drive used to have a partner. Both were inseparable – practically a single operative, until the meticulous blades and levers of counter-intelligence prised the two apart. Now one holds the virus and the other the antidote. There’s no agency more powerful than the one drawing the two back together.

Were the University of Lincoln’s Enrico Ferrari a scriptwriter, that might be how the movie would be pitched. For better or worse, Ferrari is a scientist. Rather than screenplays, there are journal articles – and a new drug delivery method for nanomedicine ready for in vivo trials. The spy is streptococcus pyogenes (S-PY), a scarcely noticeable extra in most people’s throats, but the uncharismatic villain of pharyngitis, impetigo and necrotising fascitis. In 2012, researchers at Oxford University’s Department of Biochemistry split the bacterium around its highly unusual extra covalent isopeptide bond to create SpyTag and SpyCatcher, two proteins that spontaneously conjoin when they come into contact.

For some, this could be a love story, but, from Ferrari’s perspective, SpyTag and SpyCatcher are microbiology’s USB. They form the basis for the plugand- play model for delivering nanomedicine. At the moment, the use of nanoparticles to precisely target otherwise insoluble or hard to administer treatments like chemotherapy, is an exciting area of scientific research. “But maybe in the sense of drugs on the market,” says Ferrari, “it’s not enormously successful.” The USB standard was developed to make computers easier to use for those with no experience; Ferrari’s goal is to do something similar for nanomedicine.

In the 2018 paper ‘Modular assembly of proteins on nanoparticles’, published in Nature Communications, Ferrari and the team showed that SpyCatcher/SpyTag can be used to decorate gold nanoparticles with proteins for more targeted drug delivery. Less summarily, SpyCatcher can be immobilised on gold nanoparticles with the enzyme Glutathione S-Transferase (GST) without losing any functionality, making it possible to covalently bind therapeutic proteins modified with SpyTag. “With SpyCatcher- SpyTag, you have a sort of automatic connector of proteins that works by simple mixing and forms a very solid bond,” enthuses Ferrari. “Anything that expresses SpyTag will bond to SpyCatcher, so, if SpyCatcher is already fused to GST, it means that will inevitably bring it to bind with gold. It’s the USB socket and plug that I was missing.”

This approach has a host of advantages for the gold nanoparticles that Ferrari has worked on so far, but the most exciting element is its potential to be cheaply and universally applicable for different combinations of proteins and nanomaterials, just as the original USB was for computers and peripherals.

“You have particles of different materials,” Ferrari goes on to explain. “Then you have a variety of biomolecules that scientists are trying to conjugate to nanoparticles to make them active or to make them specific for some target.”

This requires an enormous effort of optimisation to do that for every combination. “But if you are able to make a particle that will hold this USB socket, you don’t need that effort,” says Ferrari. “You just need to make a different adapter in place of GST, which instead of binding to gold will bind to iron oxide, for example. As long as your molecule has the USB plug, it will work.”

Size of a nanomaterial.
Journal of Pharmaceutical Investigation

Make the drop

Imagine one loose brick, one cut-out book, one hollow coin in a city of millions. It is used for a dead drop – a decidedly pre-cyber way for spies to exchange or transmit information without meeting. Still, it would be a key plot point for Ferrari’s USB thriller. Armed only with some vague clues about its possible whereabouts and the knowledge that it is likely to be near to some chalk hieroglyphs (which were often used to indicate that a drop had been made), one operative is tasked with finding it.

That is the best espionage-based analogy for the task of delivering nanomedicine to the right place in the body. The proteins on nanocarriers need to trigger binding events with very specific target cells so they can be absorbed by endocytosis. As such, therapeutic proteins are typically chosen and engineered for their affinity with a specific receptor on target cells.

However, what Ferrari euphemistically calls “the recognition event”, becomes much less likely if those proteins are damaged as they travel through the body. While an operative could do with some clear directions, the medicine needs what Ferrari refers to as “a clean and well-organised assembly” to ensure the interaction between the targeting protein and its target.

Just as the best spies can become double agents if they are not properly monitored and handled, the most promising nanocarriers can ‘leak’ if they are poorly assembled. To maintain an equilibrium in the bloodstream, blood serum proteins work to cover up or detach therapeutic molecules from nanoparticles in vivo, creating a ‘corona’ that can impact the medicine’s chemical make-up and disrupt its therapeutic effect. Once contact is made, those proteins also activate the body’s immune system, which will attack the nanocarrier as a potentially dangerous invader. All in all, it is exceedingly difficult to predict how this might play out in different individuals.

“That’s the problem,” says Ferrari. “It might be that the proteins that stick to the particles will overwhelm the chemistry that you originally developed on the surface, so it won’t do exactly what you intended. Even more dramatically, you might end up having particles that work well on your bench but have completely different effects in vivo, because the corona forms differently.”

But spies know how to avoid unwanted attention. These spies in particular comprise one of the very few systems for constructing complete multi-protein mega-molecules without chemical cross-linking. With GST, the pair covalently immobilise therapeutic biomolecules on gold nanoparticles, locking their chemical structure into a functional, hierarchical corona with its own equilibrium, thus preventing the dynamic exchange of molecules in the bloodstream. Whereas current approaches cross link particles to proteins without controlling the strength of their connection, Ferrari’s method allows for the best orientation of covalent bonds on both the gold-side and on the protein-side, guaranteeing that there will be no leaks.

“It is a hybrid method between chemical conjugation and passive adsorption,” Ferrari explains. “It has all the advantages of passive adsorption, which is simple mixing – easy to handle – but it also has the advantage that it provides covalent bonds between all the individual components in a layer-by-layer deposition.”

Leaks are hard to explain, but it is far from the only problem with the current nanomedical paradigm. At present, all the different materials, molecules and mixtures that constitute targeted nanomedicines need to be approved by regulators. As Ferrari fears, “Having an enormous number of combinations of elements, that you have to make use of to make a variety of different nanomedicines with different proteins, might be unsustainable, too expensive or too difficult to do.”

However, with a modular platform, the same building blocks can be established as safe to reuse with different molecules and nanocarriers. “It’s a mix-and-match procedure that may save on getting nanomedicine through regulatory bodies,” explains Ferrari, who is well aware that it is just as important as human ones.

The remarkable discovery

None of this emphasis on tiny spycraft is to underplay the importance of GST. Ferrari credits its remarkable, previously undiscovered ability to form sulphur bonds to gold and silver as making the difference in the team’s research.

Still, to fully leverage the potential of modular nanomedicine, Ferrari needs to find appropriately strong equivalents to GST for other clinically significant nanocarriers, such as silica and polymers. The challenge comes from the fact that those materials are far less reactive than gold or silver, which makes it highly unlikely that the interaction can be built around a similar covalent bond.

While alternatives are investigated, Ferrari is looking for the right pharmaceutical partner and therapeutic molecule to use with gold nanoparticles for in vivo tests and, ultimately, a full clinical trial.

That said, once Ferrari finds a way to attach the USB to materials other than precious metals, it is believed that the approach could achieve even greater universality as a conjugation method for nanoscale environmental biotechnology. The example of safely delivering a particular enzyme to contaminated soil without compromising its functionality is given: “The same way nanoparticles are used for drug delivery, passing from the syringe to the target in the human body, I think they can go from a bucket to the soil to target contaminants,” Ferrari explains.

The main difference? “If you’re going to deliver a drug into a patient maybe you can use gold, but if you have to spread it across a contaminated field – that’s probably a bit expensive.” After all the blood, the gold, the intrigue and the paranoia, who could begrudge two spies their rural retirement?