Francesco Tampieri

This study presents a novel protocol to quantify the surface distribution, penetration depth and concentration of reactive oxygen and nitrogen species (RONS) generated by plasma in biocompatible hydrogels, serving as tissue surrogates. Using colorimetric probes, we visualized and quantified RONS interaction through color intensity analysis. Two types of hydrogels were used—agarose and gelatin—at different concentrations, to study how composition and concentration affect the interaction of plasma‐generated RONS. The results show that the surface distribution and penetration depth of RONS increase with treatment time and depend on the chemical structure and concentration of the hydrogel. This methodology offers a reproducible, animal‐free approach to optimize plasma‐based therapies by enabling precise assessment of ROS and RNS behavior in 3D tissue‐like environments.

Oxidative stress‐based therapies exploit common cancer traits—elevated reactive oxygen species and altered redox metabolism,—making tumors more susceptible to oxidative damage from, e.g., cold atmospheric plasma, an ionized gas containing reactive oxygen and nitrogen species (RONS). Plasma‐treated hydrogels (PTH) emerge as vehicles for RONS delivery. Here, PTH are explored as single vehicle for the combined local delivery of RONS and Doxorubicin (Dox). Dox does not impair PTH function, and its IC50 is lowered in vitro; biological characterization and monotherapy comparison are conducted in ovo . Analysis of over 200 tumors, derived from liposarcoma SW872 or osteosarcoma 143B cell lines, reveals effects in the latter after a single PTH‐Dox administration: synergistic GPX4 reduction, with the fraction of high cytoplasmic GPX4 cells dropping from 47% to 24%, and diminished average tumor weight, with multiple tumors (of smaller initial size) weighting 70% less than control. In contrast, mono‐therapies enhanced the protective GPX4/NRF2 mRNA or protein expression. GPX4 is a ferroptosis inhibitor linked to therapy resistance. Observing effects after a single PTH‐Dox hydrogel administration is exceptional, as indirect plasma treatment has until now required frequent dosing in complex tumor models. For clinical use, multimodal approaches are key, and PTHs offer a convenient platform.

This study investigates the impact of reactive oxygen species produced by nonthermal plasma on biopolymers using computer simulations. Specifically, reactive molecular dynamics simulations are employed to study the interaction between oxygen atomsa key short-lived component generated during direct plasma treatmentand the alginate molecule, which serves as the model system in our analysis. The simulations reveal that oxygen atom impact leads to significant structural changes, including oxygen addition (44.5%), glycosidic bond cleavage (13.5%), ring opening (31%), and organic peroxide formation (25%) (these events are not mutually exclusive, and therefore, the percentages do not sum directly to 100%). Additionally, the oxidation process results in carboxyl group reduction and CO₂ detachment (13%), potentially altering the cross-linking properties of alginate. Our model results align with existing experiment findings and provide deep insight into the interaction between alginate and plasma-generated reactive species. This is fundamental for the use of biopolymers, particularly those capable of forming hydrogels, combined with plasma, for biomedical applications.

Plasma jets, crucial atmospheric pressure sources in biomedical applications, generate reactive species in liquids, with electrical fields playing a significant role, as variations in pulse rise times and durations in dielectric barrier discharges yield diverse effects. This study presents a novel nanosecond pulse plasma jet. Here, investigations with phosphate‐buffered saline and Ringer's saline elucidate critical parameters influencing species generation, such as treatment time and gas flow rate. Results showed increasing concentrations of H₂O₂ and NO₂⁻ over time, with NO₂⁻ degrading faster in Ringer's saline due to acidification. The nanosecond pulse jet exhibits superior energy efficiency than conventional jets, laying the groundwork for optimizing species generation and studying electrical field effects in future biological works.

Epithelial ovarian cancer (EOC) is the most common type of gynecological tumor, presenting poor prognosis at diagnosis and with recurrences being frequently observed. Reactive species generated by physical plasma and transferred into liquids have shown promising results in cancer therapy. Recently, Ringer's lactate solution was exposed to plasma showing selective anticancer activity on EOC cells. In this work, we compared the effect of plasma treatment, using the kINPen plasma jet, on Ringer's saline and Ringer's lactate solution. These two plasma‐treated liquids were analyzed chemically by quantifying reactive species and the extent of lactate oxidation. The biological efficiency of the plasma‐treated liquids was explored in EOC cells. The results show that lactate is affected by plasma treatment, displaying a reduction of cytotoxic potential.

Fundamental and applied research on plasma‐treated liquids for biomedical applications was boosted in the last few years, dictated by their advantages with respect to direct treatments. However, often, the lack of consistent analysis at a molecular level of these liquids, and of the processes used to produce them, have raised doubts of their usefulness in the clinic. The aim of this article is to critically discuss some basic aspects related to the use of plasma‐treated liquids in medicine, with a focus on their chemical composition. We analyze the main liquids used in the field, how they are affected by non‐thermal plasmas, and the possibility to replicate them without plasma treatment.

The mutual interaction between reactive species generated by non-thermal plasma and biopolymers in solution causes oxidative modifications that can have an impact in biomedical applications.

Hydrogels have been recently proposed as suitable materials to generate reactive oxygen and nitrogen species (RONS) upon gas-plasma treatment, and postulated as promising alternatives to conventional cancer therapies. Acting as delivery vehicles that allow a controlled release of RONS to the diseased site, plasma-treated hydrogels can overcome some of the limitations presented by plasma-treated liquids in in vivo therapies. In this work, we optimized the composition of a methylcellulose (MC) hydrogel to confer it with the ability to form a gel at physiological temperatures while remaining in the liquid phase at room temperature to allow gas-plasma treatment with suitable formation of plasma-generated RONS. MC hydrogels demonstrated the capacity for generation, prolonged storage and release of RONS. This release induced cytotoxic effects on the osteosarcoma cancer cell line MG-63, reducing its cell viability in a dose-response manner. These promising results postulate plasma-treated thermosensitive hydrogels as good candidates to provide local anticancer therapies.

Freshly squeezed apple juice was subjected to air non-thermal plasma treatment to investigate the capability of this processing method to inactivate microorganisms and to evaluate its safety when applied to liquid food products. Two different configurations of a transient spark discharge in ambient air were tested: an electrospray system with the juice flowing directly through the high voltage needle electrode, and a batch system, where the discharge was generated onto the surface of the juice. The key physico-chemical parameters of the juice, such as pH, conductivity, color, transmittance, and Brix degree, did not significantly change upon treatment. The concentration of nitrate ions formed by the plasma was safe, while that of nitrite ions and hydrogen peroxide was initially higher than the safety limits, but decreased within 24 h post treatment. The plasma effect on individual natural components of the juice, such as sugars, organic acids, and polyphenols, treated in water solutions led to their partial or substantial decomposition. However, when these compounds were plasma-treated altogether in the juice, they remained unaffected. The antimicrobial effect of the plasma processing was evaluated via the inoculation of model microorganisms. A stronger (6 log) decontamination was detected for bacteria Escherichia coli with respect to yeast Saccharomyces cerevisiae. Plasma processing led to a substantial extension of the juice shelf-life by up to 26 days if refrigerated, which represents a promising application potential in food technology.