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Atmospheric Pressure plAsma meets biomaterials for bone Cancer HEaling

Periodic Reporting for period 2 - APACHE (Atmospheric Pressure plAsma meets biomaterials for bone Cancer HEaling)

Reporting period: 2018-10-01 to 2020-03-31

Cold atmospheric pressure plasmas (APP) have been reported to selectively kill cancer cells without damaging the surrounding tissues. Studies have been conducted on a variety of cancer types but to the best of our knowledge not on any kind of bone cancer. Treatment options for bone cancer include surgery, chemotherapy, etc. and may involve the use of bone grafting biomaterials to replace the surgically removed bone.
APACHE brings a totally different and ground-breaking approach in the design of a novel therapy for bone cancer by taking advantage of the active species generated by APP in combination with biomaterials to deliver the active species locally in the diseased site. The feasibility of this approach is rooted in the evidence that the cellular effects of APP appear to strongly involve the suite of reactive species created by plasmas, which can be derived from a) direct treatment of the malignant cells by APP or b) indirect treatment of the liquid media by APP which is then put in contact with the cancer cells.
The project, if successful is important for the society as it develops a therapy that is selective (avoiding side effects) for a disease that despite being minoritary, affects essentially children and young adolescents, so positive outcomes will have a great impact.
In APACHE we aim to investigate the fundamentals involved in the lethal effects of cold plasmas on bone cancer cells, and to develop improved bone cancer therapies. To achieve this we will take advantage of the highly reactive species generated by APP in the liquid media, which we will use in an incremental strategy: i) to investigate the effects of APP treated liquid on bone cancer cells, ii) to evaluate the potential of combining APP treated liquid in a biomaterial vehicle with/wo CaP biomaterials and iii) to ascertain the potential three directional interactions between APP reactive species in liquid medium with biomaterials and with chemotherapeutic drugs.
The methodological approach involves an interdisciplinary team, dealing with plasma diagnostics in gas and liquid media; with cell biology and the effects of APP treated with bone tumor cells and its combination with biomaterials and/or with anticancer drugs.
In this first 30 months of the project, we have worked intensely on setting up the laboratory, the equipment, implementation of new experimental protocols, hiring the new research team and, in sum, to set-off the project for proper development, and have advanced significantly in different areas of the project.

From the scientific point of view, we have worked in different areas as foreseen in the DoA:
i. A more physic-chemical part related to the characterization of plasma sources and their effects in different liquids,
ii. A part related with biomaterials, where different polymers have been developed, and the effects of plasmas therein have been evaluated with regard to the generation of RONS, and
iii. A more biological-oncological area where we have been evaluating the effects of different plasma-treated liquids on a variety of cells.

Therefore, we have totally or partially achieved some of the milestones set up in the project:
- M1 & M2: Characterization of the main species of the plasma jets in different conditions, & Comparison between the characteristics of the different plasma sources employed
Optical emission spectroscopy has been employed to characterize the main species from the gas phase, as well as the rotational and vibrational temperatures of the plasma gas phase, from the 3 different plasma sources. Different conditions of distance and gas flow have been evaluated with the different sources in presence and absence of liquid media during plasma treatment.
- M3, M4: Characterization of main species generated by APP in liquids, cell culture media & with biomaterials
Different liquids (water, saline solutions), cell culture media with different compositions have been treated with plasma
- M6: Synthesis and physic-chemical characterization of biocompatible polymers
We have explored the preparation of different biocompatible polymers.Generation of RONS has been studied for the different biomaterials, and their potential modifications have been characterized by relevant techniques (FTIR, SEM, Rheology) .
- M7: Synthesis & characterization of CaP biomimetic microspheres & their combination with the biomaterials
This item is currently ongoing, and we are working in the development of different formats of calcium phosphates that will be in a second stage incorporated to the biomaterials.
- M9: Determination of the stability and lifetime of APP-liquid generated species blended in the biomaterials
This has been achieved for some of the biomaterials developed and characterized under plasma treatment up to now, and is under investigation for others.
- M11: Correlation of [ROS & RNS] in liquids and biomaterials with their anticancer potential
We have been able to correlate the amount of RONS in different liquids (in particular cell culture media and some saline solutions) on their effects on bone cancer cells. As a general rule, higher amounts of RONS are related with bone cell toxicity, and we have already been able to intuit a threshold for toxicity or survival for healthy bone cells, so we’re moving in the right direction to ascertain suitable dose.
- M12: Fundamental description of the lethal effects of APP-treated liquids & vehicles on bone cancer cells
We are currently working on the description of the possible mechanisms involved in the bone anticancer effects shown by plasma jet treated liquids, as observed by alterations in mitochondria, metabolic activity, protein expression etc.
It is expected that the project will allow to progress beyond the state of the art in bone osteosarcoma-related therapies, as we have already obtained proof of concept of selective bone anticancer activity from plasma-treated cell culture media, and saline solutions. We are currently obtaining similar in vitro results with other liquid vehicles , and quickly progressing to 3D tumor models to be able to evaluate situations closer to the real scenario.
The plasma-treated biomaterials we are currently developing and characterizing will allow a further step forward in this sense.
Atmospheric pressure Plasma Jet treatment of liquids in the ERC APACHE project