Cold Atmospheric Pressure Plasmas (CAPs) for Skin Wound Healing

In the past 20 years, cold atmospheric pressure plasmas (CAPs) have become a new promising way for many biomedical applications, such as disinfection, cancer treatment, root canal treatment, wound healing, and other medical applications. Among these applications, investigations of plasma for skin wound healing has gained huge success both in vitro and in vivo experiments, and also the mechanism behind it has been studied by many groups. In this chapter, we summarize the state-of-the-art progress in wound healing by CAPs. The plasma devices developed for wound healing, the interactions between plasmas and microorganisms/cells/tissues, the in vitro and in vivo treatments, the clinical trials, and biosafety issues are all included

Plasma-induced death of HepG2 cancer cells: Intracellular effects of reactive species

Reports show that cold atmospheric-pressure plasmas can induce death of cancer cells in several minutes. However, very little is presently known about the mechanism of the plasma-induced death of cancer cells. In this paper, an atmospheric-pressure plasma plume is used to treat HepG2 cells. The experimental results show that the plasma can effectively control the intracellular concentrations of ROS, NO and lipid peroxide. It is shown that these concentrations are directly related to the mechanism of the HepG2 death, which involves several stages. First, the plasma generates NO species, which increases the NO concentration in the extracellular medium. Second, the intracellular NO concentration is increased due to the NO diffusion from the medium. Third, an increase in the intracellular NO concentration leads to the increase of the intracellular ROS concentration. Fourth, the increased oxidative stress results in more effective lipid peroxidation and consequently, cell injury. The combined action of NO, ROS and lipid peroxide species eventually results in the HepG2 cell death. The mechanism of death of human hepatocellular carcinoma cells (HepG2) induced by atmospheric-pressure room-temperature plasma, related to the plasma-controlled intracellular concentrations of reactive oxygen species (ROS), nitric oxide (NO) and lipid peroxide is revealed. Only 34.75 s are required to reduce the number of the viable HepG2 cells by 50%

APRTP-Js treatment disturbed intracellular calcium homeostasis.

<p>Cells were treated without or with APRTP-Js for 240 s, 480 s and 720 s, respectively. After continuous culturing for 24 h, the cells were loaded with calcium probe Fluo-3/AM and the intracellular free Ca²⁺ was measured using a FACScan flow cytometer.</p

APRTP-Js treatment induced apoptosis in HepG2 cells.

<p>Cells were treated by APRTP-Js for 240 s, 480 s and 720 s, and then cultured for 24 h. (A) Cells were double stained with annexin V-FITC and PI and analyzed by flow cytometry. Cells that stained positive for annexin V-FITC and negative for PI were undergoing early stage of apoptosis; Cells that stained positive for both annexin V-FITC and PI were in the end stage of apoptosis; Cells that stained negative for both annexin V-FITC and PI were alive and not undergoing measurable apoptosis. Percentage of apoptotic cells (annexin V-FITC positive) was shown by histogram. (B) Observation of Hoechst 33342 apoptosis staining by fluorescence microscopy. Examples of typical apoptotic nuclei were indicated by white arrows.</p

Typical emission spectra of the plasma emitted by APRTP-Js.

<p>(A) 275-500 nm (B) 500-800 nm.</p

NAC attenuated the apoptosis induced by APRTP-Js treatment.

<p>(A) HepG2 cells were incubated for 1h with 10 mM NAC (N+) or cell culture medium (N-), followed by treatment with the indicated dose of plasma. Apoptosis was measured 24 h after APRTP-Js treatment by collecting and staining the cells with annexin V-FITC/PI. Samples were run on the flow cytometry. P- N-: control group without any treatment; P+ 240s N-: cells with 240 s plasma exposure only; P+ 480s N-: cells with 480 s plasma exposure only; P-N+: cells with NAC pre-treatment only; P+ 240s N+: cells with NAC pre-treatment followed by 240 s plasma exposure; P+ 480s N+: cells with NAC pre-treatment followed by 480 s plasma exposure. (B) Percentage of apoptotic cells was determined from flow cytometry results.</p

Schematic of APRTP-Js used in the HepG2 cell treatment.

<p>(A) Schematic of the APRTP-Js device and (B) photograph of APRTP-Js acting on the HepG2 cells.</p

APRTP-Js treatment induced oxidative and nitrative damage in HepG2 cells.

<p>(A) Status of total protein nitration. The total nitration statuses of proteins were studied using a nitrotyrosine antibody by Western blot in cells with or without APRTP-Js treatment. The deepened nitrotyrosine epitopes were indicated by arrows. (B) Status of protein carbonyl content. The protein carbonyl content was measured using a commercial assay kit. Values are means ± SEM obtained from three independent experiments. The asterisk represents statistical significance in comparison with control (*p < 0.05 and **p < 0.01).</p

APRTP-Js Treatment compromised cellular antioxidant defense system in HepG2 cells.

<p>Cells were treated without or with APRTP-Js for 240 s, 480 s and 720 s, respectively. 24 h later, the cells were harvested and lysed using cell lysis buffer. The samples were centrifuged and the supernatants were used for antioxidant activities assay. (A) The effect of APRTP-Js treatment on total GSH content in HepG2 cells. (B) The effect of APRTP-Js treatment on the SOD activity in HepG2 cells. (C) The effect of APRTP-Js treatment on the catalase activity in HepG2 cells. Values are means ± SEM obtained from three independent experiments. The asterisk represents statistical significance in comparison with control value (*p < 0.05 and **p < 0.01).</p