Periodic Reporting for period 4 - APACHE (Atmospheric Pressure plAsma meets biomaterials for bone Cancer HEaling)
Reporting period: 2021-10-01 to 2022-12-31
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.
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.
During the APACHE project, we have worked intensely and have been able to set up a laboratory in Plasma Medicine, unique in our country for the moment, we have developed and implemented new experimental protocols, established a research team and, most importantly, have been able to accomplish the different objectives set in the project, leading to breakthroughs in the field, and opening of new research areas.
From the scientific point of view, we have worked in the different areas 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 a) different polymers in solution have been selected, and the effects of plasmas therein have been evaluated with regard to the generation of RONS, and b)
iii. A more biological-oncological area where we have evaluated the effects of different plasma-treated liquids and hydrogels on a variety of osteosarcoma cells, on 3D models that we have developed within the project, and also in ex-vivo and in vivo models.
The work performed during the development of the project has allowed to reach all 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
- M3, M4 & M5: Characterization of main species generated by APP in liquids (water, saline solutions), cell culture media & biomaterials (hydrogels and calcium phosphates)
- M6: Synthesis and physic-chemical characterization of 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 & M8: Synthesis & characterization of CaP biomimetic microspheres & their combination with the biomaterials, and Anticancer drug loading and release from CaP microspheres and from CaP microspheres + hydrogels
- M9 & M10: Determination of the stability and lifetime of APP-liquid generated species blended in the biomaterials
- M11: Correlation of [ROS & RNS] in liquids and biomaterials with their anticancer potential. 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, which is key for selective therapy in absence of harmful side effects.
- M12: Fundamental description of the lethal effects of APP-treated liquids & vehicles on bone cancer cells. We have made great progress on the description of the possible mechanisms involved in the anticancer effects shown by plasma jet treated liquids on osteosarcoma, as observed by alterations in mitochondria, metabolic activity, protein expression etc.
- M13, M14 : Determination of osteoconductivity of biomaterials APP-liquid-loaded in orthotopic in vivo models & Determination of the antiproliferative efficiency of the APP-liquid based novel therapies designed
The IP generated in the project has been protected with patents, and the results published in high impact journals like Advanced science, Nature Protocols, Free Radical Biology and Medicine, Biomaterials Science, Analytical chemistry, ACS Appl. Mater. Interfaces, etc.
We have also disseminated the results in diferent academic and scientific forums through invited and contributed oral communications, and also more general dissemination to the public through videos, interviews, etc.
From the scientific point of view, we have worked in the different areas 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 a) different polymers in solution have been selected, and the effects of plasmas therein have been evaluated with regard to the generation of RONS, and b)
iii. A more biological-oncological area where we have evaluated the effects of different plasma-treated liquids and hydrogels on a variety of osteosarcoma cells, on 3D models that we have developed within the project, and also in ex-vivo and in vivo models.
The work performed during the development of the project has allowed to reach all 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
- M3, M4 & M5: Characterization of main species generated by APP in liquids (water, saline solutions), cell culture media & biomaterials (hydrogels and calcium phosphates)
- M6: Synthesis and physic-chemical characterization of 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 & M8: Synthesis & characterization of CaP biomimetic microspheres & their combination with the biomaterials, and Anticancer drug loading and release from CaP microspheres and from CaP microspheres + hydrogels
- M9 & M10: Determination of the stability and lifetime of APP-liquid generated species blended in the biomaterials
- M11: Correlation of [ROS & RNS] in liquids and biomaterials with their anticancer potential. 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, which is key for selective therapy in absence of harmful side effects.
- M12: Fundamental description of the lethal effects of APP-treated liquids & vehicles on bone cancer cells. We have made great progress on the description of the possible mechanisms involved in the anticancer effects shown by plasma jet treated liquids on osteosarcoma, as observed by alterations in mitochondria, metabolic activity, protein expression etc.
- M13, M14 : Determination of osteoconductivity of biomaterials APP-liquid-loaded in orthotopic in vivo models & Determination of the antiproliferative efficiency of the APP-liquid based novel therapies designed
The IP generated in the project has been protected with patents, and the results published in high impact journals like Advanced science, Nature Protocols, Free Radical Biology and Medicine, Biomaterials Science, Analytical chemistry, ACS Appl. Mater. Interfaces, etc.
We have also disseminated the results in diferent academic and scientific forums through invited and contributed oral communications, and also more general dissemination to the public through videos, interviews, etc.
The project has lead to progress beyond the state of the art, in different areas, given its highly interdisciplinary character. We have obtained proof of concept of selective bone anticancer activity from plasma-treated cell culture media, saline solutions and hydrogels. This has been demonstrated through different 2D, 3D, ex vivo and in vivo models for relevant results. We have developed composite biomaterials for bone cancer therapy, shown their in vivo selectivity, and have made significant advances both in mechanistic aspects of the anticancer action of pasma-treated liquids and biomaterials, as well as on methodological and technical developments of relevance to the field, especially with regard to homogeneisation in the field of Plasma Medicine, as well in what regards determination and quantification of reactive species from plasmas in different vehicles.