Analysis: 'Training' nanoparticles to target tumours

Researchers have used two common characteristics of cancer cells to help nanoparticles home in on them and, if developed successfully for medical use, show up tumours much more clearly on MRI scans than has been possible so far

The nanoparticles work by ‘sticking’ to cancer cells and agglomerating to clumps large enough to improve the contrast of that region compared with normal tissue on a MRI scan.

The intensity of pixels on the image depends largely on the magnetic relaxation time – the time it takes for the spins to become disordered after they have been artificially aligned by an external magnetic field. The most commonly-used particles to date rely on organometallic complexes based on the rare earth element gadolinium that are designed to pass into the human bloodstream. A typical application is to use the complexes to see if the blood-brain barrier has been reached – as the gadolinium will be picked up in regions where it should not register.

“Some doctors feel that, even though MRI scanners are effective at spotting large tumours, they are perhaps not as good at detecting smaller tumours in the early stages,” explains team leader Professor Nicholas Long from the Department of Chemistry at Imperial College London.

In recent years, attention has turned to iron-oxide nanoparticles as they could prove to be far more chemically versatile. These are particles that are small enough to no longer behave ferro-magnetically. Instead, they are what is called super paramagnetic, becoming magnetised in the presence of strong magnetic fields – such as those encountered in an MRI scanner – but then losing it when the field disappears. They boost contrast by interfering with the weak local magnetic fields formed by hydrogen nuclei.

The configuration of hydrogen atoms within biomolecules creates small differences in local magnetic fields that the MRI picks up. Iron-oxide nanoparticles interfere with these fields and speed up the rate at which their spins fall back out of alignment when the RF pulse used by the machine to point them in the same direction has finished. As a result, the regions with a larger concentration of nanoparticles look much darker than regular tissue.

Some simple iron-oxide nanoparticles are readily taken up by organs such as the liver, spleen, and bone marrow. “Iron oxide nanoparticles have been around for a while even in hospitals,” says Juan Gallo, researcher in the department of surgery and cancer at Imperial College London, “but they are not targetted.”

By attaching them to biomolecules, however, it becomes possible to make the nanoparticles much more specific, to the point where they will latch onto tumour cells. There is no single chemical marker that will distinguish a cancerous cell from a normal one. Being the result of random genetic mutations, cancers can be incredibly diverse. A recent analysis of breast cancers found that even the most commonly mutated or commonly deleted genes occurred in just 10 per cent of affected patents. “There is a long tail of other mutations, each affecting just a few per cent or less of patients,” says Peter Campbell, head of cancer genetics and genomics at the Wellcome Trust Sanger Institute.

However, tumours in specific organs often follow similar patterns of behaviour. They tend to have mutations that suppress genes that would otherwise regulate their ability to grow and spread. For example, many cancers overproduce a class of metal-based enzymes that are associated with healing wounds.

Normally, cells around wounds produce a series of materials that help bind them together and grow over the damage, using matrix metalloproteases to form the membranes. Tumours produce some of these metalloproteases in large quantities because they have lost the ability to control their manufacture. The Imperial College team, in common with others working in these area, chose not just to focus on two of these – MMP2 and MMP9 – but also on a receptor protein that appears in cell walls to provide an anchor for the nanoparticles.

Nanoparticles can be tagged with short strands of protein that bind to the anchor – a molecule called CXCR4 that, like the MMPs, is often over-produced by cancer cells. As a result, the nanoparticles are more likely to attach to cancerous cells than to healthy ones. The relatively small basic nanoparticles do not show up well on an MRI, however.

Larger particles show a more dramatic effect. But if introduced to the body in too large a form they are less likely to reach the target because they are more likely to be cleared from the bloodstream by white blood cells. They also can prove toxic if too large. This is where the MMPs come in – they break off protecting chemical chains so that the nanoparticles can bind with one another in a further chemical reaction.

“Aggregation also changes and improves their magnetic properties as MRI contrast agents,” says Gallo. “There were a few attempts in the past by other groups to do this, but their final aggregation was not based on a controlled chemical reaction, so it was more dependent on physical properties that are difficult to control or predict in vivo.”

For the binding reaction, the Imperial team harnessed a relatively recently introduced form of chemistry named for its combination of near-ideal properties in which the reagents more or less ‘click’ together without forming unwanted by-products or demanding high temperatures to activate. Click chemistry reagents are also designed not to react with the many biomolecules in the body and are based on chemical groups that do not appear in most living organisms, such as triple-bonded carbon groups and azides, short chains or rings of nitrogen atoms.

Early forms of click chemistry demanded a copper catalyst, which ruled them out of use in the body; but more recently chemists have found variants that use the strain inherent in their structure to help the reaction along. In the Imperial experiment, when the protein on the nanoparticle’s surface binds with MMP2 or MMP9, it exposes the azide and alkyne groups that also coat it, allowing them to react and bond with similarly attached particles. The result is a larger clump that shows up more clearly under MRI.

“The innovation of our work is the combination of all these pieces to create a probe that not only localises and accumulates in the tumours, but it also responds to it, changing its nature,” Gallo points out. “Copper-free click chemistry is quite new but has already been used pre-clinically in animal models.”

In the technique developed by the Imperial team, there is no reaction to shut off the nanoparticles’ tendency to clump together once they reach a certain size.

Gallo adds: “There is, in theory, no limit to the size the aggregated particles can get to. This worried us a bit in the beginning as large aggregates could block capillaries and small veins. In practice, when you inject something into the blood stream it is diluted down in the blood, and circulates until it is cleared out – or in our case until it accumulates in the tumour.”

Gallo adds: “This circulation makes it more difficult for the probes to make these big aggregates – they are not all of them present in the same place at the same time. In our in vivo tests we didn't lose any animal before the end of the experiments.”

The group is now looking at making further changes to the nanoparticles such as adding fluorescent markers that are activated selectively by cancerous cells so that they show up better using other forms of medical imaging.

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