Plasma therapy for cancer: A new approach for delivery of active agents without radiation beams

What is gas plasma medicine and how can it be used to treat cancer? 

According to the Institute of Health and Welfare (2019)¹¹, one in two Australians will be affected by cancer by the age of 85. Of those affected, 50% would benefit from radiation treatment (Figure 1).  The use of ionizing radiation is a well-established and effective therapy in cancer treatment. Radiation therapy uses high energy beams, which penetrate healthy tissue to target the tumour. The ionizing radiation kills cancer by damaging the cell function or its DNA. Damage is caused directly by the radiation and indirectly by producing free radicals that then go on to disrupt molecules in and around the cells. An estimated two-thirds of damage caused by radiation treatment is thought to be through the indirect effect (Hall et al., 2006)⁴.

Conventional radiotherapy has the ability to deliver a prescribed radiation dose with high accuracy to a known volume and this is particularly effective for solid and well-defined tumours. However, not all tumours are solid. Mesothelioma for example is a cancer that grows in the mesothelium, the membrane surrounding the lungs. The spread of mesothelioma makes it difficult to target this cancer with a radiation beam without causing unwanted damage to the lungs and other vital healthy tissues. For such cancers, it would be beneficial to introduce the active components of radiation therapy into the mesothelial layer, without using high energy X-ray or electron beams. One way we could do this is by creating free radicals in the solution using a gas plasma treatment and then introducing the activated solution into the target site to be treated.

What is gas plasma? 

Gas plasma is produced by exposing gas to strong electric fields.  Electrons are stripped from the gas molecules, which gives the gas plasma special properties. Such a supercharged gas becomes electrically conductive; it gives off light and is capable of producing a range of free radical species. These reactive species, which are created when the free electrons from the plasma collide with molecules in the air or in liquid, are known as ‘Reactive Oxygen and Nitrogen Species’, or RONS. The RONS  are the same reactive species as those created by conventional ionising radiation beams. They are responsible for the indirect cell damage caused in conventional radiation therapy and are described above (Zhang and Martin, 2014¹⁸). 

Unlike high energy radiotherapy beams, the RONS in a gas plasma are the sole active components. They do not have the accompanying damaging direct effects of high energy radiation. Therefore, the risk of gas plasma harm to surrounding healthy tissue, when compared to conventional radiotherapy, may be reduced. 

In some parts of the United States and Europe, gas plasma devices have been registered for the treatment of patients with chronic wound infections, dermatological issues; and in surgical applications.  Some of these registered devices are under investigation for the treatment of head and neck tumours (Metelmann et al., 2015⁸ and Schuster et al., 2016¹³). However, the current generation of gas plasma generators is not suited for all clinical applications in cancer therapy. As they are not designed for the activation of solutions for cancer treatment, improved methods of generating gas plasma-activated solutions for cancer treatment need further research and development. Here we describe how the active components of gas plasma, the RONS, are used to activate a liquid for cancer treatment.

Gas plasma-activated solutions 

Gas plasma-activated solutions are created when gas plasma is generated over the surface of a liquid (such as water or cell culture medium) and the RONS travel through the air and diffuse into the liquid via a process that can be described by Brownian motion (more on this later). 

The activated liquid is used to treat cancer, either in vitro in a flask or in vivo in a mouse. However, more studies are needed to better understand the many ways in which the activated solution kills the cancer cells; and how effective and safe the treatment would be for humans. There are reports in the literature describing gas plasma-activated solutions selectively killing cancer cells at doses that do not harm healthy normal cells (Mohades et al., 2016⁹ and Tanaka et al., 2011¹⁵). Despite the fact that the preclinical data for the efficacy of plasma-treated solutions for cancer treatment is still in its very early stages, it is very encouraging so far. There are a few early animal studies that indicate plasma-activated solutions are capable of reducing the tumour burden in mice (Liedtke et al., 2017 ⁷ and Hattori et al., 2015⁵ and Takeda et al., 2017¹⁴). Increased life-span of the treated mice, compared to the untreated mice, was shown by Nakamura et al., 2017¹⁰. It has been suggested that plasma treatment may even be able to prevent pancreatic metastases and gastric cancers (Sato et al., 2018¹²) which currently have very poor prognoses.

The existing evidence indicates that gas plasma-treated solutions do not have a negative effect on major organs such as the heart, lung and liver in mice (Dehui et al., 2018¹). It should be noted that the Dehui study only studied the effects of plasma-treated solution on normal tissue in healthy mice with no cancer. Although gas plasma-activated solutions at this stage appear to offer a promising new treatment, more investigations are needed to ensure the technique is safe and effective for the treatment of humans. 

The administration of RONS in the form of a solution offers potential benefits. Unlike radiotherapy, where increasing the effective voltage (i.e. energy) of an X-ray or electron beam allows the beam to penetrate deeper into the body,  increasing the energy applied to the plasma does not increase the penetration of the dose into the body in the same way. 

Similarly, when the reactive species are administered by direct exposure to gas plasma, rather than in a solution, they are not able to penetrate deeply into tissue.  Duan et al. (2017²) reported penetration of up to 1.25 mm through muscle tissue, which may be enough to get through to the dermal layers (Wei et al 2017¹⁶) but is certainly not enough to get through to cancers deeper in the body. 

Our own simulation research suggests that the distances reactive molecules are able to travel in the water may be even less than that (explained more below). If a gas plasma-treated liquid can be injected directly into a tumour or cavity (such as the mesothelial or abdominal cavity) it will enable the reactive species to act directly where it is needed and overcome the penetration issue.

Currently, to increase the dose (i.e. the concentration of RONS) administered to a patient and/or liquid, exposure time to gas plasma must be increased. Long treatment times are inefficient and can become uncomfortable for patients. But if the solution can be successfully treated prior to injection, by loading the solution with sufficient RONs, the treatment time can be minimised and the patient’s comfort can be improved. 

Characterisation of the gas plasma pen 

The aim of using the gas plasma treatment device (referred to as ‘gas plasma pen’) described in this work is: 

•  to create small volumes of patient specific gas plasma-activated solutions; and

• to investigate further the anti-cancer capability of this solution. 

The gas plasma pen consists of a narrow glass tube, which contains an insulated wire passing down the centre and an external hollow electrode wrapped around the far end of the glass tube. The central wire acts as the high-voltage electrode. A  high voltage AC power supply was used to deliver to the gas plasma pen 7.5 kV at a frequency of approximately 20 kHz. High purity argon was used as the treatment gas, which flows through the glass tube surrounding the central electrode. The strong electric fields in the argon gas at atmospheric pressure, facilitate ignition of the plasma. It is seen as the purple glowing plume in the Figure below.

The RONS production by the gas plasma pen was determined in a pilot study by using a  solution of methylene blue dissolved in water. Methylene blue bleaches in the presence of OH^. and H₂O₂ radicals above a certain concentration (Huang et al., 2010⁶) which enables the level of RONS production to be determined. The bleaching of the methylene blue is a surrogate measure of the RONS produced. From these measurements (Figure 6), we were able to improve the design of the gas plasma pen for the best performance.

The gas plasma treatment resulted in a significant reduction in absorbance of the methylene blue solution after 2 minutes of treatment time. The control was a ‘sham’ exposure, where argon gas was allowed to flow over the methylene blue solution for 2 minutes without gas plasma present. The sham experiment showed increased absorbance compared to the stock solution. The reduction in absorbance indicates that plasma treatment has bleached the methylene blue solution, and therefore, confirms the production of hydroxyl ions in the gas plasma. The increase in absorbance during sham treatment confirms that the argon plays no part in the bleaching of the methylene blue. This was most likely caused by evaporation of the water increasing the methylene blue concentration under sham exposure conditions.  

This simple test ensures that the solution is being loaded with reactive species. It also provides important information for optimising the treatment parameters such as: 

• treatment distance; and 
• treatment volume 

to understand the rate of change of RONS concentration in the solution. It’s important to ensure that the RONS produced by the plasma is transferred quickly and efficiently to the treatment solution because an increased treatment distance (the distance between the surface of the solution and the plasma plume) has been shown to be less effective at killing cells (Wende et al., 2015¹⁷).  The final configuration, based on our plasma pen device, still requires optimisation. But we are confident that this device will become a useful tool to generate gas plasma-treated solutions for cancer treatment. One such feature requiring optimisation is the need for the whole system to remain completely sterile – a vital requirement in cell culture experiments and for future clinical implementation.

Random walk simulation

Once the free radicals in the solution are delivered to the treatment site, the range of treatment effects needs to be evaluated. To visualise the activation process of the solution via the plasma pen and to quantitatively determine the distribution of radicals in water, simulation results were obtained. The gas plasma produces many different types of reactive species. For simplicity, the simulation examined the diffusion in the water of five of the most well-understood species: 

• hydroxyl radicals;
• superoxide; 
• nitrite ions; 
• nitric oxide; and 
• hydrogen peroxide 

when affected by Brownian motion.

Brownian motion occurs due to the random motion of particles suspended in a liquid. The particles bump into each other and into other water molecules, which causes them to move around. The phenomenon was first described by botanist Robert Brown and mathematically described by Einstein (Einstein et al., 1905³). This is an important concept for consideration during the characterisation of a plasma pen device because it’s the reactive species that are generated and propagated in the liquid that provide the anti-cancer properties. The simulations were used to determine the distance that the radical molecules are able to travel through the water during their half-lives, assuming no reactions occurred during this time.

From Figure 8, it can be seen that superoxide, nitrite ions and hydroxyl radicals have much shorter half-lives and thus can be found much closer to their points of origin than hydrogen peroxide or nitric oxide which have much longer half-lives. 

The results showed that: 

• the hydroxyl radical is the most reactive species examined in this experiment and can reasonably be expected to move only a few nanometres during its half-life. 

•  Superoxide and nitric oxide move a little further – approximately 0.3 and 0.2 micrometres respectively. 

• Hydrogen peroxide and nitrite ions have the longest half-lives, enabling them to move up to 0.6 mm each.  

These distances are not large, which means that the created plasma radicals cannot travel far into the body. As mentioned earlier, this limits the ability of direct gas plasma treatment for tumours deeper in the body. But a gas plasma-activated solution may be able to overcome these issues and will be able to deliver the treatment locally at the target site.  

This simulation is the first step towards a more complete understanding of the distribution of radical species in a solution activated by our gas plasma device. 

Conclusion 

Radiation therapy is a cornerstone of contemporary cancer treatment. An active component of this treatment is through free radicals in the indirect effect. Here we propose a way of delivering only the free radicals in a solution via our gas plasma pen device. This is a promising approach because it offers the same active cancer-killing components as radiation therapy, without the unwanted normal tissue damage of ionizing radiation beams. Since the plasma produced radicals are unable to diffuse far in water and do not penetrate far in tissue, gas plasma-activated solutions injected into the target site may offer highly targeted and effective cancer treatment.  

For more on gas plasma and gas plasma-activated solutions, please see my article:

Cancer treatment with gas plasma and with gas plasma-activated liquid: positives, potentials and problems of clinical translation

Acknowledgements

Thank you to graphic designer Maria Mora for her fantastic work on Figure 2. Thank you to Professor David McKenzie and Associate Professor Natalka Suchowerska for everything.

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Juliette C Harley 20 July 2021