Multi-Modal Biological Destruction by Cold Atmospheric Plasma: Capability and Mechanism

Abstract

Cold atmospheric plasma (CAP) is a near-room-temperature, partially ionized gas composed of reactive neutral and charged species. CAP also generates physical factors, including ultraviolet (UV) radiation and thermal and electromagnetic (EM) effects. Studies over the past decade demonstrated that CAP could effectively induce death in a wide range of cell types, from mammalian to bacterial cells. Viruses can also be inactivated by a CAP treatment. The CAP-triggered cell-death types mainly include apoptosis, necrosis, and autophagy-associated cell death. Cell death and virus inactivation triggered by CAP are the foundation of the emerging medical applications of CAP, including cancer therapy, sterilization, and wound healing. Here, we systematically analyze the entire picture of multi-modal biological destruction by CAP treatment and their underlying mechanisms based on the latest discoveries particularly the physical effects on cancer cells.

Keywords: cancer therapy; cell death; cold atmospheric plasma; microorganism sterilization; virus inactivation.

Figure 2

A general picture of plasma medicine. (a) Schematic illustration of the biological effect of CAP. Here, biofilm was used as an example [26]. (b) Growth inhibition of CAP source (DBD) on bacteria (S. Typhimurium) grown on LB agar plates: control (left) and DBD treatment (right) [31]. (c) Confocal scanning laser microscopic imaging of strong bacterial death in a CAP-treated biofilm grown on polycarbonate coupons. Live cells and dead cells were present in green and red, respectively [30]. (d) The improved wound healing of the inflamed ulcer on a human leg by CAP treatment [39]. (e) Tumor inhibition effect of CAP jet treatment on subcutaneously xenografted bladder tumor on mice [8]. (f) The inactivation of feline calicivirus and the inhibited infectivity on its host Crandell-Reese feline kidney cells by CAP treatment [48].

Figure 1

Typical CAP sources. (a) A volume DBD source [16,17]. (b) A surface DBD source [18]. (c) A CAP jet source. The length of plasma jet can be modulated by controlling signals, such as pulse width (µs) [14].

Figure 3

A typical observation of apoptosis in the CAP-treated melanoma cells B16F10. (a) Cellular images of cytochrome c release by immunocytochemistry and fluorescence-activated cell sorting analysis to check mitochondria membrane potential change. (b) Western blot analysis of the phosphorylation on serine 139 on H2AX histone (γ-H2AX), p53, and caspase-3. (c) Western blot analysis of BAX and BCL-2 [18].

Figure 4

CAP induces oxidative stress response in malignant mesothelioma cells (SM2), resulting in increased endocytosis, lysosome biogenesis, and autophagy-associated cell death. (a) Schematic autophagy mechanism of the CAP-treated malignant mesothelioma cells. (b) TEM imaging of vesicle structures, endosome-like vesicles, and organelles involved in the autophagic pathway of the CAP-treated SM2 cells (60 s). Yellow and red arrowhead indicates AP and AL, respectively. Scale bar = 500 nm. (c) Fluorescent imaging of the formation of autophagosomes in the CAP-treated (60 s) malignant mesothelioma cells. Cells were stained by DAPI (DNA, blue), LC3B (red), and LAMP1 (green). LC3B suggested the presence of autophagosomes, while the colocalization of LC3 and LAMP1 suggests the autophagolysosome formation (yellow overlay fluorescence). Scale bar = 20 μm. ***, p < 0.001 [104].

Figure 5

Direct observation of necrosis on the physically based CAP-treated melanoma cells (B16F10). (a) Schematic illustration. (b) A time-lapse observation of bubbling. (c) The fluorescent imaging of the treated B16F10 cells. Microtubules (green) and DNA (blue) were stained using BioTracker 488 green microtubule cytoskeleton dye and Hoechst 33342, respectively [20].

Figure 6

The cell death of bacteria after CAP treatment. (a) The interplay of physical destruction and biological cell death upon CAP treatment. (b) False-colored SEM images of bacteria exposed to CAP with high discharge voltage (HV) and low discharge voltage (LV) [149].

Figure 7

The CAP-triggered damage on T4 bacteriophage. (a) Schematic illustration. (b) TEM-negative stained imaging of T4 bacteriophage treated by CAP and the CAP-treated water [156].

Figure 8

The CAP-triggered damage on FCV. (a) TEM imaging of calicivirus the control (left) and the experimental group (15s, right). Blue arrows, black arrows, and red arrows refer to untreated virus particles, distorted viral particles, and the debris of the damaged viral particles, respectively. (b) The calculated location of oxidized peptide residues (yellow colored) among the N-terminal arm (NTA) domain (I); shell (S) domain (II); P1 subdomain (III); and P2 subdomain (IV). (c) The principles of the quantification of capsid destruction due to CAP treatment. Reverse transcription PCR (RT-PCR) was used to quantify the unaffected calicivirus’s RNA. (d) The agarose gel (DNA) patterns of EMA-coupled RT-PCR products from viral RNA obtained from control, 15 s, and 120 s of CAP treatment [48].

Figure 9

SARS-CoV-2 structure. (a) Molecular architecture of SARS-CoV-2 virus obtained by cryoelectron tomography and subtomogram averaging. The S protein in “RBD down” conformation, “one RBD up” conformation, lipid envelope, and ribonucleoproteins (RNPs) are shown in salmon, red, gray, and yellow, respectively [160]. (b) The cryoelectron microscopy structure of RBD-ACE2 complex [162].

Figure 10

A schematic illustration of typical CAP treatment on cancer cells and bacteria in vitro. In most experimental setups in vitro, cancer cells or mammalian cells were immersed in a layer of medium during direct CAP treatment. In contrast, many bacterial cells were directly exposed to CAP because solid culture medium was widely used in many cases. Such a different experimental tradition may naturally filter the physical effectors of CAP in the studies involving liquid culture medium.