For almost as long as doctors have known about X-rays, they have been using them to treat cancer. Although Emil Grubbe pioneered radiotherapy after observing its damaging effects on his skin, he thought that damage was superficial, something cleared up with little more than petroleum jelly and time. By contrast, chemotherapy did not figure in treating the same conditions until its associated dangers were indisputable. It took the horror wrought by chemical weapons in the First World War, and the study of their effects in the Second World War, for scientists to realise the breakthrough chemical treatments heralded in their ongoing battle with cancer.

Now, researchers at Australia’s ARC Centre of Excellence for Nanoscale BioPhotonics have developed a method to deliver chemotherapy drugs with ‘X-ray-triggerable liposomes’, combining the two treatments in a way that can increase their effectiveness and minimise their side effects.

“Our method makes it possible to perfectly synchronise both treatments so they can be given simultaneously,” explains Ewa Goldys, a senior researcher on the project. “This enables enhanced therapeutic outcomes with potentially reduced doses of drug and/or radiation required because of this exquisitely precise timing of drug release.”

As the team’s research paper – ‘Controlled gene and drug release from a liposomal delivery platform triggered by X-ray radiation’, published in Nature Communications – rather less elegantly recommends, “From a clinical point of view, it would be beneficial to have access to this multimodality treatment, given our evidence of better therapeutic effect (or, potentially, equal therapeutic effect) at diminished toxicity, in the case when single modality treatment options alone can only produce desired therapeutic effects at a significant cost of short and long-term toxicity.”

The key ingredients

Injectable liposomes are the most established nanomaterial vehicles for targeted drug delivery. As the name indicates, their aqueous core is surrounded by a lipid bilayer, which facilitates cellular uptake of the liposome’s content due to its similarity to cell membranes. Biocompatible, biodegradable and able to isolate hydrophilic and hydrophobic drugs from the surrounding environment, liposomes are extremely well-qualified drug delivery systems. In fact, one of their few limitations is the lack of tuneable triggers they offer to control drug release. That is precisely the issue this research solves.

“We have ensured that the liposomes release their drug payload at exactly the right time and in exactly the right place to ensure the most effective treatment,” explains Dr Wei Deng.

Whereas previous triggering approaches have made liposomes sensitive to changes in pH, externally applied heat, or light, Deng’s team attuned their liposomes to react to X-ray radiation using gold nanoparticles and the photosensitive molecule verteporfin (VP).

“The radiation from the X-ray causes the verteporfin to react and to produce highly reactive singlet oxygen (1O2) that then destabilises the liposomal membrane, causing the release of the drug,” Deng explains. “The gold nanoparticles are added into the mix as they focus the X-ray energy. This enhances the singlet oxygen generation and hence improves the speed of the membrane break-up.”

More specifically, gold reacts so strongly with X-rays that gold nanoparticles are highly effective ‘radiosensitisers’ able to amplify the radiation doses in tumour tissue. As the paper highlights, these nanoparticles can also “selectively scatter or absorb the high-energy X-ray radiation, leading to enhanced energy transfer from X-ray to photosensitisers. With such contribution, the VP molecules in the presence of gold nanoparticles are able to interact more strongly with ionising radiation than the VP on its own, causing enhanced 1O2 generation”. As such, the team’s tests demonstrate that liposomes ‘doped’ with gold nanoparticles and VP molecules aid drug delivery by generating by far the highest amounts of destabilising 1O2 under X-ray, approximately 79% more than unenhanced liposomes.

Target cancer

So much for the mechanism; all that 1O2 generation is only relevant as far as it impacts treatment. As Deng indicated above, the destabilising effect works to release an antisense oligonucleotide for gene silencing and the chemotherapy drug doxorubicin (Dox) from inside the liposome (LipoDox). When used for in-vitro testing, the effects were dramatic. Without X-ray triggering, the LipoDox was found to kill about 10% of human colorectal cancer cells. When it was triggered, however, that proportion rose to 50%. Equally encouraging is the fact that assessments of X-ray-induced damage in genetic materials indicated that the levels of X-ray radiation and doxorubicin did not cause obvious damage to DNA molecules.

Similarly, once released, the antisense oligonucleotide prevented the translation of the PAC1R mRNA by blocking the translation initiation complex, thus helping to stop the cancer spreading. Its effects were less pronounced – but still notable. 24 hours after X-ray exposure, the density of PAC1R decreased by around 45%, compared with a 30% decrease in cells that received received the liposomes without X-ray triggering. If anything, the results of in vivo tests were even more impressive. Whereas bowel tumours in mice treated with liposomes and X-rays grew by 2.9-fold and 3.4-fold respectively in the two weeks after treatment, in the group treated with X-ray-triggered liposomes the tumours actually shrunk. There was a 74% reduction in tumour volume compared with the control group, and the X-ray-triggered liposome treatment also achieved the largest amount of tumour necrosis.

Moreover, no mortality was observed during 14 days after treatment with X-ray-triggered liposomes, and no weight loss of treated mice was observed compared with the control, suggesting that this combined technique is well tolerated by mice under the present conditions. If she was not a doctor, Goldys’ assertion that this is an extremely encouraging result might be considered an understatement.

Minimise side effects

Of course, there are reasons for the research team to remain level-headed – no cancer treatment is without its side effects. It took time, but the pioneers of X-ray gradually came to realise that the radiation they worked with had the potential not merely to fight the condition, but cause it. The first related death took place nine years after X-rays were discovered. Charles Madison Dalley was Thomas Edison’s glassblower. Both of his arms were amputated in a futile attempt to stop his cancer spreading before it killed him at 39.

In 1951, the radiotherapy pioneer Grubbe was asked to move out by his landlord: he had been so disfigured by X-ray-related facial tumours that he was scaring away other tenants. The side effects of chemotherapy are less ghoulish, but we are all aware of the difficulty of dealing with the hair loss, exhaustion, nausea and vomiting, to name just a few of its debilitating impacts.

In the case of X-ray-triggerable liposomes, one of the most troubling possible dangers is the fact that simultaneous chemo and radiotherapy may result in damage to the heart. This fact prompted the team to test the same approach using the chemotherapy drug etoposide (ETP), which is associated with reduced incidence of cardiotoxicity, instead of Dox. These tests also indicate that chemotherapy drugs are more effective when triggered with X-ray radiation.

Similarly, the harmful effects of singlet oxygen – the primary cytotoxic agent released as part of this drug delivery technique – are limited by its short lifespan, which means it cannot travel beyond a very limited area. On top of that, the reaction by which the 1O2 destabilises liposome membranes consumes 1O2 radicals, further minimising any adverse effects.

Although the common use of VP, lipids, Dox and X-rays in the treatment of tumours suggests the strategy of using X-ray-triggerable liposomes to deliver drugs should translate well to the clinical context, gold nanoparticles are not yet approved by all regulatory agencies. Even so, the study notes that the size of gold nanoparticles is compatible with the requirements of renal clearance, which means that longterm nanoparticle toxicity is likely to be minimised, if not eliminated, as the body flushes them out. Tumour-specific targeting ligands could also be grafted onto the particles, ensuring they interact with the tumour without coming into contact with the rest of the body. In fact, the paper makes clear that “the ease of conjugation of targeting ligands to liposome surface with appropriate linkers would be an added advantage when applied to the targeted therapy, in particular for tumour treatment”.

Future modalities

The team is now working to further optimise the X-ray-triggered liposomes while they scale up their processes and develop the clinical protocols necessary to get clinical approval for their first in-human trials.

As with almost all cancer treatments, it feels like that approval can’t come soon enough. An estimated 9.6 million people will die from cancer in 2018. By 2030, that number is predicted to reach 13 million. Even more monumentally, statistics suggest that half of all people in the UK will get cancer in their lifetime. Appropriately, then, the crux of Deng and Goldys’ work is that combinatory cancer treatments have a better therapeutic effect than singlemodality approaches, as they make it possible to cure or manage cancers while doing far less damage to the patient’s quality of life. Beyond that, however, their approach shows an elegant genius in finding a specific meeting point for two modalities. Humanity has come a long way from the hideous, tragic beginnings of chemotherapy and X-ray use.