Does non-thermal plasma modify biopolymers in solution? A chemical and mechanistic study for alginate.

In the last decades, non-thermal plasma has been extensively investigated as a relevant tool for various biomedical applications, ranging from tissue decontamination to regeneration and from skin treatment to tumor therapies. This high versatility is due to the different kinds and amount of reactive oxygen and nitrogen species that can be generated during a plasma treatment and put in contact with the biological target. Some recent studies report that solutions of biopolymers with the ability to generate hydrogels, when treated with plasma, can enhance the generation of reactive species and influence their stability, resulting thus in the ideal media for indirect treatments of biological targets. The direct effects of the plasma treatment on the structure of biopolymers in water solution, as well as the chemical mechanisms responsible for the enhanced generation of RONS, are not yet fully understood. In this study, we aim at filling this gap by investigating, on the one hand, the nature and extent of the modifications induced by plasma treatment in alginate solutions, and, on the other hand, at using this information to explain the mechanisms responsible for the enhanced generation of reactive species as a consequence of the treatment. The approach we use is twofold: (i) investigating the effects of plasma treatment on alginate solutions, by size exclusion chromatography, rheology and scanning electron microscopy and (ii) study of a molecular model (glucuronate) sharing its chemical structure, by chromatography coupled with mass spectrometry and by molecular dynamics simulations. Our results point out the active role of the biopolymer chemistry during direct plasma treatment. Short-lived reactive species, such as OH radicals and O atoms, can modify the polymer structure, affecting its functional groups and causing partial fragmentation. Some of these chemical modifications, like the generation of organic peroxide, are likely responsible for the secondary generation of long-lived reactive species such as hydrogen peroxide and nitrite ions. This is relevant in view of using biocompatible hydrogels as vehicles for storage and delivery reactive species for targeted therapies.

Thermosensitive hydrogels to deliver reactive species generated by cold atmospheric plasma: a case study with methylcellulose.

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.

Selectivity of direct plasma treatment and plasma-conditioned media in bone cancer cell lines.

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.

Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures.

Cold atmospheric plasma (CAP) is a potential anticancer therapy. CAP has cytotoxic effects when applied either directly to cancer cell cultures or indirectly through plasma-conditioned liquids. This protocol describes how to treat adherent cultures of human cancer cell lines with CAP or plasma-conditioned medium and determine cell viability following treatment. The protocol also includes details on how to quantify the reactive oxygen and nitrogen species present in medium following CAP treatment, using chemical probes using UV-visible or fluorescence spectroscopy. CAP treatment takes ~30 min, and 3 h are required to complete quantification of reactive oxygen and nitrogen species. By providing a standardized protocol for evaluation of the effects of CAP and plasma-conditioned medium, we hope to facilitate the comparison and interpretation of results seen across different laboratories.

Enhanced Generation of Reactive Species by Cold Plasma in Gelatin Solutions for Selective Cancer Cell Death.

Atmospheric pressure plasma jets generate reactive oxygen and nitrogen species (RONS) in liquids and biological media, which find application in the new area of plasma medicine. These plasma-treated liquids were demonstrated recently to possess selective properties on killing cancer cells and attracted attention toward new plasma-based cancer therapies. These allow for local delivery by injection in the tumor but can be quickly washed away by body fluids. By confining these RONS in a suitable biocompatible delivery system, great perspectives can be opened in the design of novel biomaterials aimed for cancer therapies. Gelatin solutions are evaluated here to store RONS generated by atmospheric pressure plasma jets, and their release properties are evaluated. The concentration of RONS was studied in 2% gelatin as a function of different plasma parameters (treatment time, nozzle distance, and gas flow) with two different plasma jets. Much higher production of reactive species (H2O2 and NO2-) was revealed in the polymer solution than in water after plasma treatment. The amount of RONS generated in gelatin is greatly improved with respect to water, with concentrations of H2O2 and NO2- between 2 and 12 times higher for the longest plasma treatments. Plasma-treated gelatin exhibited the release of these RONS to a liquid media, which induced an effective killing of bone cancer cells. Indeed, in vitro studies on the sarcoma osteogenic (SaOS-2) cell line exposed to plasma-treated gelatin led to time-dependent increasing cytotoxicity with the longer plasma treatment time of gelatin. While the SaOS-2 cell viability decreased to 12%-23% after 72 h for cells exposed to 3 min of treated gelatin, the viability of healthy cells (hMSC) was preserved (∼90%), establishing the selectivity of the plasma-treated gelatin on cancer cells. This sets the basis for designing improved hydrogels with high capacity to deliver RONS locally to tumors.

Positive Effect of Cold Atmospheric Nitrogen Plasma on the Behavior of Mesenchymal Stem Cells Cultured on a Bone Scaffold Containing Iron Oxide-Loaded Silica Nanoparticles Catalyst.

Low-temperature atmospheric pressure plasma was demonstrated to have an ability to generate different reactive oxygen and nitrogen species (RONS), showing wide biological actions. Within this study, mesoporous silica nanoparticles (NPs) and FexOy/NPs catalysts were produced and embedded in the polysaccharide matrix of chitosan/curdlan/hydroxyapatite biomaterial. Then, basic physicochemical and structural characterization of the NPs and biomaterials was performed. The primary aim of this work was to evaluate the impact of the combined action of cold nitrogen plasma and the materials produced on proliferation and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (ADSCs), which were seeded onto the bone scaffolds containing NPs or FexOy/NPs catalysts. Incorporation of catalysts into the structure of the biomaterial was expected to enhance the formation of plasma-induced RONS, thereby improving stem cell behavior. The results obtained clearly demonstrated that short-time (16s) exposure of ADSCs to nitrogen plasma accelerated proliferation of cells grown on the biomaterial containing FexOy/NPs catalysts and increased osteocalcin production by the cells cultured on the scaffold containing pure NPs. Plasma activation of FexOy/NPs-loaded biomaterial resulted in the formation of appropriate amounts of oxygen-based reactive species that had positive impact on stem cell proliferation and at the same time did not negatively affect their osteogenic differentiation. Therefore, plasma-activated FexOy/NPs-loaded biomaterial is characterized by improved biocompatibility and has great clinical potential to be used in regenerative medicine applications to improve bone healing process.

Production of reactive species in alginate hydrogels for cold atmospheric plasma-based therapies.

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.

Pyruvate Plays a Main Role in the Antitumoral Selectivity of Cold Atmospheric Plasma in Osteosarcoma.

Osteosarcoma (OS) is the most common primary bone tumor but current therapies still have poor prognosis. Cold Atmospheric Plasma (CAP) and Plasma activated media (PAM) have shown potential to eliminate cancer cells in other tumors. It is thought that Reactive Oxygen and Nitrogen species (RONS) in PAM are key players but cell culture media composition alters treatment outcomes and data interpretation due to scavenging of certain RONS. In this work, an atmospheric pressure plasma jet was employed to obtain PAM in the presence or absence of pyruvate and used to treat the SaOS-2 (OS) cell line or hBM-MSC healthy cells. OS cells show higher sensitivity to PAM treatment than healthy cells, both in medium with and without pyruvate, activating apoptosis, DNA damage and deregulating cellular pathways mediated by c-JUN, AKT, AMPK or STAT3. In line with previous works, lack of pyruvate increases cytotoxic potential of PAM affecting cancer and healthy cells by increasing 10-100 times the concentration of H2O2 without altering that of nitrites and thus decreasing CAP anti-tumor selectivity. Suitable conditions for CAP anti-cancer selectivity can be obtained by modifying plasma process parameters (distance, flow, treatment time) to obtain adequate balance of the different RONS in cell culture media.

Tyrosinase-Loaded Multicompartment Microreactor toward Melanoma Depletion.

Melanoma is malignant skin cancer occurring with increasing prevalence with no effective treatment. A unique feature of melanoma cells is that they require higher concentrations of ltyrosine (l-tyr) for expansion than normal cells. As such, it has been demonstrated that dietary l-tyr restriction lowers systemic l-tyr and suppresses melanoma advancement in mice. Unfortunately, this diet is not well tolerated by humans. An alternative approach to impede melanoma progression will be to administer the enzyme tyrosinase (TYR), which converts l-tyr into melanin. Herein, a multicompartment carrier consisting of a polymer shell entrapping thousands of liposomes is employed to act as a microreactor depleting l-tyr in the presence of melanoma cells. It is shown that the TYR enzyme can be incorporated within the liposomal subunits with preserved catalytic activity. Aiming to mimic the dynamic environment at the tumor site, l-tyr conversion is conducted by co-culturing melanoma cells and microreactors in a microfluidic setup with applied intratumor shear stress. It is demonstrated that the microreactors are concurrently depleting l-tyr, which translates into inhibited melanoma cell growth. Thus, the first microreactor where the depletion of a substrate translates into antitumor properties in vitro is reported.

Multicompartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells.

Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles. In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct subcompartments. Compartmentalization allows for multiple processes to take place in close vicinity and in a parallel manner without the risk of interference or degradation. Here, we report on a subcompartmentalized and intracellularly active carrier, a crucial step for advancing artificial organelles. In particular, we develop and characterize a novel capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell.

Intracellular Microreactors as Artificial Organelles to Conduct Multiple Enzymatic Reactions Simultaneously.

The creation of artificial organelles is a new paradigm in medical therapy that aims to substitute for missing cellular function by replenishing a specific cellular task. Artificial organelles tackle the challenge of mimicking metabolism, which is the set of chemical reactions that occur within a cell, mainly catalyzed by enzymes. So far, the few reported carriers able to conduct enzymatic reactions intracellularly are based on single-compartment carriers. However, cell organelles outperform by conducting multiple reactions simultaneously within confined sub-compartments. Here, the field of artificial organelles is advanced by reporting the assembly of a microreactor consisting of polymer capsules entrapping gold nanoclusters (AuNCs) and liposomes as sub-compartments. The fluorescence properties of AuNCs are employed to monitor the microreactors uptake by macrophages. Encapsulation is demonstrated and functionality of microreactors with trypsin (TRP) and horseradish peroxidase (HRP)-loaded liposomes is preserved. Multiple enzymatic reactions taking place simultaneously is demonstrated by exposing macrophages with the internalized microreactors to bis-(benzyloxycarbonyl-Ile-Pro-Arg)-Rho-110 and Amplex Red substrates, which are specific for TRP and HRP, respectively. Conversion of the substrates into the respective fluorescent products is observed. This report on the first microreactor conducting multiple enzymatic reactions simultaneously inside a cell is a considerable step in the field of artificial organelles.