BACKGROUND: Current therapies for bone cancers -either primary or metastatic -are

In the last years, great advances have been made in therapies based in cold atmospheric plasmas (CAP). CAP generate reactive oxygen and nitrogen species (RONS) which can be transferred to liquids. These CAP activated liquids display the same biological efficacy (i.e. on killing cancer cells) as CAP themselves, opening the door for minimally invasive therapies. However, injection of a liquid in the body results in fast diffusion due to extracellular fluids and blood flow. Therefore, the development of efficient vehicles which allow local confinement and delivery of RONS to the diseased site is a fundamental requirement. In this work, we investigate the generation of RONS (H2O2, NO2−, short-lived RONS) in alginate hydrogels by comparing two atmospheric pressure plasma jets: kINPen and a helium needle, at a range of plasma treatment conditions (time, gas flow, distance to the sample). The physic-chemical properties of the hydrogels remain unchanged by the plasma treatment, while the hydrogel shows several-fold larger capacity for generation of RONS than a typical isotonic saline solution. Part of the RONS are quickly released to a receptor media, so special attention has to be put on the design of hydrogels with in-situ crosslinking. Remarkably, the hydrogels show capacity for sustained release of the RONS. The plasma-treated hydrogels remain fully biocompatible (due the fact that the species generated by plasma are previously washed away), indicating that no cytotoxic modifications have occurred on the polymer. Moreover, the RONS generated in alginate solutions showed cytotoxic potential towards bone cancer cells. These results open the door for the use of hydrogel-based biomaterials in CAP-associated therapies.

Atmospheric pressure plasma jets have been shown to impact several cancer cell lines, both in vitro and in vivo. These effects are based on the biochemistry of the reactive oxygen and nitrogen species generated by plasmas in physiological liquids, referred to as plasma-conditioned liquids. Plasma-conditioned media are efficient in the generation of reactive species, inducing selective cancer cell death. However, the concentration of reactive species generated by plasma in the cell culture media of different cell types can be highly variable, complicating the ability to draw precise conclusions due to the differential sensitivity of different cells to reactive species. Here, we compared the effects of direct and indirect plasma treatment on non-malignant bone cells (hOBs and hMSCs) and bone cancer cells (SaOs-2s and MG63s) by treating the cells directly or exposing them to previously treated cell culture medium. Biological effects were correlated with the concentrations of reactive species generated in the liquid. A linear increase in reactive species in the cell culture medium was observed with increased plasma treatment time independent of the volume treated. Values up to 700 µM for H2O2 and 140 µM of NO2− were attained in 2 mL after 15 min of plasma treatment in AdvDMEM cell culture media. Selectivity towards bone cancer cells was observed after both direct and indirect plasma treatments, leading to a decrease in bone cancer cell viability at 72 h to 30% for the longest plasma treatment times while maintaining the survival of non-malignant cells. Therefore, plasma-conditioned media may represent the basis for a potentially novel non-invasive technique for bone cancer therapy.

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Current bone cancer treatments include and may combine surgery, chemotherapy, radiation therapy or cryosurgery among others. However, these treatments are not completely effective and unspecific where patients relapse. In recent years, researchers have found a novel approach by means of cold atmospheric plasma that can selectively treat cancer cells without damaging the surrounding tissues but its effects on bone cancer are unknown. Cold plasmas generate reactive oxygen and nitrogen species in air, which seem to be related to their effects on cells. As biological tissues and cells are covered with biological fluids during plasma treatment, studies point out an implication of the liquid media in the effects of plasmas. Within this context, in this research project the effect of an atmospheric pressure plasma jet will be investigated in two different liquid media by implementing appropriate measurement protocols for three different reactive species: [H 2 O 2 ]; [HO • ] and [NO 2-]. To ascertain its biological effects this atmospheric pressure plasma jet treated liquids will be put in contact with bone cancer cells (SaOs-2) and also with healthy bone cells (Hobs). Preliminary results show that the atmospheric pressure plasma jet treated liquids contain different amounts of the studied reactive species depending on the treatment conditions and the medium employed, and can selectively kill bone cancer cells while conserving the viability of healthy bone cells.

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MassJCharge 3.SD.01 3-00 400 ' Figure 1. MS spectra of cysteine solutions (300 µM) acquired before (A) and after plasma treatment (B). Treatment conditions: 5 minutes, 3 slm Ar/N2/ 02 feed gas (99-0.5-0.5%).