Dose-Dependent Effects in Plasma Oncotherapy: Critical In Vivo Immune Responses Missed by In Vitro Studies.

Cold atmospheric plasma (CAP) generates abundant reactive oxygen and nitrogen species (ROS and RNS, respectively) which can induce apoptosis, necrosis, and other biological responses in tumor cells. However, the frequently observed different biological responses to in vitro and in vivo CAP treatments remain poorly understood. Here, we reveal and explain plasma-generated ROS/RNS doses and immune system-related responses in a focused case study of the interactions of CAP with colon cancer cells in vitro and with the corresponding tumor in vivo. Plasma controls the biological activities of MC38 murine colon cancer cells and the involved tumor-infiltrating lymphocytes (TILs). In vitro CAP treatment causes necrosis and apoptosis in MC38 cells, which is dependent on the generated doses of intracellular and extracellular ROS/RNS. However, in vivo CAP treatment for 14 days decreases the proportion and number of tumor-infiltrating CD8+T cells while increasing PD-L1 and PD-1 expression in the tumors and the TILs, which promotes tumor growth in the studied C57BL/6 mice. Furthermore, the ROS/RNS levels in the tumor interstitial fluid of the CAP-treated mice are significantly lower than those in the MC38 cell culture supernatant. The results indicate that low doses of ROS/RNS derived from in vivo CAP treatment may activate the PD-1/PD-L1 signaling pathway in the tumor microenvironment and lead to the undesired tumor immune escape. Collectively, these results suggest the crucial role of the effect of doses of plasma-generated ROS and RNS, which are generally different in in vitro and in vivo treatments, and also suggest that appropriate dose adjustments are required upon translation to real-world plasma oncotherapy.

Air Plasma-Activated Medium Evokes a Death-Associated Perinuclear Mitochondrial Clustering.

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy, which eliminates cancers effectively and safely, is the current clinical choice. Since aggressive tumors are substantially resistant to multidisciplinary therapies that target apoptosis, tumor-specific activation of another cell death modality is a promising avenue for meeting this goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can kill OS and OC by causing a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) based on its characteristic unipolar mitochondrial perinuclear accumulation. The APAM caused apoptotic and nonapoptotic cell death. The APAM increased mitochondrial ROS (mROS) and cell death, and the antioxidants such as N-acetylcysteine (NAC) prevented them. MPMC occurred following mitochondrial fragmentation, which coincided with nuclear damages. MPMC was accompanied by mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC, Ferrostatin-1, and Nocodazole. In contrast, the APAM induced minimal cell death, mROS generation, mLPO accumulation, and MPMC in fibroblasts. These results suggest that MPMC occurs in a tumor-specific manner via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC induction might serve as a promising target for exerting tumor-specific cytotoxicity.

Combined Effect of Cold Atmospheric Plasma and Curcumin in Melanoma Cancer.

Curcumin (CUR) has interesting properties to cure cancer. Cold atmospheric plasma (CAP) is also an emerging biomedical technique that has great potential for cancer treatment. Therefore, the combined effect of CAP and CUR on inducing cytotoxicity and apoptosis of melanoma cancer cells might be promising. Here, we investigated the combined effects of CAP and CUR on cytotoxicity and apoptosis in B16-F10 melanoma cancer cells compared to L929 normal cells using MTT method, acridine orange/ethidium bromide fluorescence microscopic assay, and Annexin V/PI flow cytometry. In addition, the activation of apoptosis pathways was evaluated using BCL2, BAX, and Caspase-3 (CASP3) gene expression and ratio of BAX to BCL2 (BAX/BCL2). Finally, in silico study was performed to suggest the molecular mechanism of this combination therapy on melanoma cancer. Results showed that although combination therapy with CUR and CAP has cytotoxic and apoptotic effects on cancer cells, it did not improve apoptosis rate in melanoma B16-F10 cancer cells compared to monotherapy with CAP or CUR. In addition, evaluation of gene expression in cancer cell line confirmed that CUR and CAP concomitant treatment did not enhance the expression of apoptotic genes. In silico analysis of docked model suggested that CUR blocks aquaporin- (AQP-) 1 channel and prevents penetration of CAP-induced ROS into the cells. In conclusion, combination therapy with CAP and CUR does not improve the anticancer effect of each alone.

Toxicity Assessment of Long-Term Exposure to Non-Thermal Plasma Activated Water in Mice.

Non-thermal plasma activated water (PAW) has recently emerged as a powerful antimicrobial agent. Despite numerous potential bio-medical applications, studies concerning toxicity in live animals, especially after long-term exposure, are scarce. Our study aimed to assess the effects of long-term watering with PAW on the health of CD1 mice. PAW was prepared from distilled water with a GlidArc reactor according to a previously published protocol. The pH was 2.78. The mice received PAW (experimental group) or tap water (control group) daily for 90 days as the sole water source. After 90 days, the following investigations were performed on the euthanatized animals: gross necropsy, teeth mineral composition, histopathology, immunohistochemistry, hematology, blood biochemistry, methemoglobin level and cytokine profile. Mice tolerated PAW very well and no adverse effects were observed during the entire period of the experiment. Histopathological examination of the organs and tissues did not reveal any structural changes. Moreover, the expression of proliferation markers PCNA and Ki67 has not been identified in the epithelium of the upper digestive tract, indicating the absence of any pre- or neoplastic transformations. The results of our study demonstrated that long-term exposure to PAW caused no toxic effects and could be used as oral antiseptic solution in dental medicine.

An In Situ FTIR Study of DBD Plasma Parameters for Accelerated Germination of Arabidopsis thaliana Seeds.

Current agricultural practices are not sustainable; however, the non-thermal plasma treatment of seeds may be an eco-friendly alternative to alter macroscopic plant growth parameters. Despite numerous successful results of plasma-seed treatments reported in the literature, the plasma-treatment parameters required to improve plant growth remain elusive due to the plethora of physical, chemical, and biological variables. In this study, we investigate the optimal conditions in our surface dielectric barrier discharge (SDBD) setup, using a parametric study, and attempt to understand relevant species in the plasma treatment using in situ Fourier transform infrared (FTIR) absorption spectroscopy. Our results suggest that treatment time and voltage are key parameters for accelerated germination; however, no clear conclusion on causative agents can be drawn.

Repeated exposure of the oral mucosa over 12 months with cold plasma is not carcinogenic in mice.

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Implementation of a Non-Thermal Atmospheric Pressure Plasma for Eradication of Plant Pathogens from a Surface of Economically Important Seeds.

Plant pathogenic bacteria cause significant economic losses in the global food production sector. To secure an adequate amount of high-quality nutrition for the growing human population, novel approaches need to be undertaken to combat plant disease-causing agents. As the currently available methods to eliminate bacterial phytopathogens are scarce, we evaluated the effectiveness and mechanism of action of a non-thermal atmospheric pressure plasma (NTAPP). It was ignited from a dielectric barrier discharge (DBD) operation in a plasma pencil, and applied for the first time for eradication of Dickeya and Pectobacterium spp., inoculated either on glass spheres or mung bean seeds. Furthermore, the impact of the DBD exposure on mung bean seeds germination and seedlings growth was estimated. The observed bacterial inactivation rates exceeded 3.07 logs. The two-minute DBD exposure stimulated by 3-4% the germination rate of mung bean seeds and by 13.4% subsequent early growth of the seedlings. On the contrary, a detrimental action of the four-minute DBD subjection on seed germination and early growth of the sprouts was noted shortly after the treatment. However, this effect was no longer observed or reduced to 9.7% after the 96 h incubation period. Due to the application of optical emission spectrometry (OES), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM), we found that the generated reactive oxygen and nitrogen species (RONS), i.e., N2, N2+, NO, OH, NH, and O, probably led to the denaturation and aggregation of DNA, proteins, and ribosomes. Furthermore, the cellular membrane disrupted, leading to an outflow of the cytoplasm from the DBD-exposed cells. This study suggests the potential applicability of NTAPPs as eco-friendly and innovative plant protection methods.

Plasma Robot Engineering: The Next Generation of Precision Disease Management.

Robotics, once combined with cold atmospheric plasma, represent key elements of the next generation of personalized medicine and contribute to the effective yet immediate response to pandemics. Plasma robots can serve as CAP delivery vehicle to assist in tumor therapeutics and viral disease prevention in addition to the already prevalent utilities of robots in precision surgery, diagnosis, and risk prevention. Plasma robots may develop at either the macro- or the micro- scale, successful navigations at which require joint effort from multiple research domains.

Inactivation and sensitization of Pseudomonas aeruginosa by microplasma jet array for treating otitis media.

Otitis media (OM), known as a middle ear infection, is the leading cause of antibiotic prescriptions for children. With wide-spread use of antibiotics in OM, resistance to antibiotics continues to decrease the efficacy of the treatment. Furthermore, as the presence of a middle ear biofilm has contributed to this reduced susceptibility to antimicrobials, effective interventions are necessary. A miniaturized 3D-printed microplasma jet array has been developed to inactivate Pseudomonas aeruginosa, a common bacterial strain associated with OM. The experiments demonstrate the disruption of planktonic and biofilm P. aeruginosa by long-lived molecular species generated by microplasma, as well as the synergy of combining microplasma treatment with antibiotic therapy. In addition, a middle ear phantom model was developed with an excised rat eardrum to investigate the antimicrobial effects of microplasma on bacteria located behind the eardrum, as in a patient-relevant setup. These results suggest the potential for microplasma as a new treatment paradigm for OM.

Tolerability of Six Months Indirect Cold (Physical) Plasma Treatment of the Scalp for Hair Loss.

Androgenetic alopecia (AGA) is a chronic form of hair loss. Cold atmospheric (physical) plasma (CAP) is partly ionized gas with various widely researched effects on living tissues. CAP is an emerging treatment modality in dermatology with uses for chronic leg ulcer, actinic keratosis, warts, and other applications. Its previously demonstrated ability to induce stem cell differentiation in various cell types makes CAP a possible treatment option for AGA. Directly creating CAP on the scalp surface has drawbacks, but indirect CAP treatment—when a CAP-treated liquid is used as topical therapy—offers an alternative. In a clinical pilot study, we treated 14 patients with AGA using the indirect CAP method for three months (4 patients) and six months (10 patients). The indirect CAP treatment was well tolerated and while the primary goal of the study was not to assess efficacy, most patients reported improvement, and the investigator’s assessment also showed improvement in most patients. Our findings create the foundation for longer, extensive trials to systematically assess the efficacy of indirect CAP treatment for AGA. ClinicalTrials.gov: NCT04379752 J Drugs Dermatol. 2020;19(12): doi:10.36849/JDD.2020.5186.

Cold Atmospheric Pressure Plasma (CAP) as a New Tool for the Management of Vulva Cancer and Vulvar Premalignant Lesions in Gynaecological Oncology.

Vulvar cancer (VC) is a specific form of malignancy accounting for 5-6% of all gynaecologic malignancies. Although VC occurs most commonly in women after 60 years of age, disease incidence has risen progressively in premenopausal women in recent decades. VC demonstrates particular features requiring well-adapted therapeutic approaches to avoid potential treatment-related complications. Significant improvements in disease-free survival and overall survival rates for patients diagnosed with post-stage I disease have been achieved by implementing a combination therapy consisting of radical surgical resection, systemic chemotherapy and/or radiotherapy. Achieving local control remains challenging. However, mostly due to specific anatomical conditions, the need for comprehensive surgical reconstruction and frequent post-operative healing complications. Novel therapeutic tools better adapted to VC particularities are essential for improving individual outcomes. To this end, cold atmospheric plasma (CAP) treatment is a promising option for VC, and is particularly appropriate for the local treatment of dysplastic lesions, early intraepithelial cancer, and invasive tumours. In addition, CAP also helps reduce inflammatory complications and improve wound healing. The application of CAP may realise either directly or indirectly utilising nanoparticle technologies. CAP has demonstrated remarkable treatment benefits for several malignant conditions, and has created new medical fields, such as "plasma medicine" and "plasma oncology". This article highlights the benefits of CAP for the treatment of VC, VC pre-stages, and postsurgical wound complications. There has not yet been a published report of CAP on vulvar cancer cells, and so this review summarises the progress made in gynaecological oncology and in other cancers, and promotes an important, understudied area for future research. The paradigm shift from reactive to predictive, preventive and personalised medical approaches in overall VC management is also considered.

Cold Atmospheric Plasma Is a Potent Tool to Improve Chemotherapy in Melanoma In Vitro and In Vivo.

Malignant melanoma is a devastating disease. Because of its aggressiveness, it also serves as a model tumor for investigating novel therapeutic avenues. In recent years, scientific evidence has shown that cold atmospheric plasma (CAP) might be a promising modality in cancer therapy. In this study, we aimed to evaluate the effect of CAP generated by an argon plasma jet alone or in combination with dacarbazine (DAC) on melanoma cells in vitro and in vivo. The effects of the CAP on inducing lipid peroxidation and nitric oxide production were higher in B16 melanoma cells in comparison to non-malignant L929 cells. Assays on cell growth, apoptosis, and expression of genes related to, e.g., autophagic processes, showed CAP to have a substantial impact in melanoma cells while there were only minoreffects in L929 cells. In vivo, both CAP monotherapy and combination with DAC significantly decreased tumor growth. These results suggest that CAP not only selectively induces cell death in melanoma but also holds promises in combination with chemotherapy that might lead to improved tumor control.

Cold atmospheric plasma as an effective method to treat diabetic foot ulcers: A randomized clinical trial.

Cold atmospheric plasma (CAP) was shown to decrease bacterial load in chronic wounds. It was also presented as a novel approach to healing wounds in both in vitro and in vivo experiments. We aimed to examine the first randomized clinical trial for the use of CAP in diabetic foot ulcers. Patients (n = 44) were randomly double-blinded, and assigned to receive standard care (SC, n = 22) without or with CAP, to be applied three times a week for three consecutive weeks (SC + CAP, n = 22), using block randomization with mixing block sizes of four. The trial was conducted at the Diabetes Research Center in Tehran, Iran. CAP was generated from ionized helium gas in ambient air, and driven by a high voltage (10 kV) and high frequency (6 kHz) power supply. Primary outcomes were wound size, number of cases reaching wound size of <0.5, and a bacterial load after over three weeks of treatment. CAP treatment effectively reduced the fraction of wound size (p = 0.02). After three weeks, the wounds to reach fraction wound size of ≤0.5 was significantly greater in the SC + CAP group (77.3%) compared to the SC group (36.4%) (p = 0.006). The mean fraction of bacterial load counted in each session 'after CAP exposure' was significantly less than 'before exposure' measures. CAP can be an efficient method to accelerate wound healing in diabetic foot ulcers, with immediate antiseptic effects that do not seem to last long.

Transdermal cold atmospheric plasma-mediated immune checkpoint blockade therapy.

Despite the promise of immune checkpoint blockade (ICB) therapy against cancer, challenges associated with low objective response rates and severe systemic side effects still remain and limit its clinical applications. Here, we described a cold atmospheric plasma (CAP)-mediated ICB therapy integrated with microneedles (MN) for the transdermal delivery of ICB. We found that a hollow-structured MN (hMN) patch facilitates the transportation of CAP through the skin, causing tumor cell death. The release of tumor-associated antigens then promotes the maturation of dendritic cells in the tumor-draining lymph nodes, subsequently initiating T cell-mediated immune response. Anti-programmed death-ligand 1 antibody (aPDL1), an immune checkpoint inhibitor, released from the MN patch further augments the antitumor immunity. Our findings indicate that the proposed transdermal combined CAP and ICB therapy can inhibit the tumor growth of both primary tumors and distant tumors, prolonging the survival of tumor-bearing mice.

Plasma Permeabilization of Human Excised Full-Thickness Skin by µs- and ns-pulsed DBD.

INTRODUCTION: Cold atmospheric plasma (CAP) is gaining increasing importance as a medical or cosmetic treatment for various indications. The technology is best suited to the treatment of surfaces such as the skin and is already used in wound care and, in exemplary case studies, the reduction of superficial tumors. Several plasma sources have been reported to affect the skin barrier function and potentially enable drug delivery across or into plasma-treated skin. OBJECTIVE: In this study, this effect was quantified for different plasma sources in order to elucidate the influence of voltage rise time, pulse duration, and power density in treatments of full-thickness skin. METHODS: We compared three different dielectric barrier discharges (DBDs) as to their permeabilization efficiency using Franz diffusion cell permeation experiments and measurements of the transepithelial electrical resistance (TEER) with full-thickness human excised skin. RESULTS: We found a significant reduction of the TEER for all three plasma sources. Permeation of the hydrophilic sodium fluorescein molecule was enhanced by a factor of 11.7 (low power) to 41.6 (high power) through µs-pulsed DBD-treated skin. A smaller effect was observed after treatment with the ns-pulsed DBD. CONCLUSIONS: The direct treatment of excised human full-thickness skin with CAP, specifically a DBD, can lead to pore formation and enhances transdermal transport of sodium fluorescein.

Non-thermal atmospheric plasma treatment of onychomycosis in an in vitro human nail model.

BACKGROUND: Onychomycosis affects almost 6% of the world population. Topical azoles and systemic antifungal agents are of low efficacy and can have undesirable side effects. An effective, non-invasive therapy for onychomycosis is an unmet clinical need. OBJECTIVE: Determine the efficacy threshold of non-thermal atmospheric plasma (NTAP) to treat onychomycosis in an in vitro model. METHODS: A novel toe/nail-plate model using cadaver nails and agarose media inoculated with Candida albicans was exposed to a range of NTAP doses. RESULTS: Direct exposure of C albicans and Trichophyton mentagrophytes to 12 minutes of NTAP results in complete killing at doses of 39 and 15 kPulses, respectively. Onset of reduced viability of C albicans to NTAP treatment through the nail plate occurs at 64 kPulses with 10× and 100× reduction at 212 and 550 kPulses, respectively. CONCLUSIONS: NTAP is an effective, non-invasive therapeutic approach to onychomycosis that should be evaluated in a clinical setting.

Cold atmospheric plasma modulates endothelial nitric oxide synthase signalling and enhances burn wound neovascularisation.

Treatment with cold atmospheric plasma (CAP) has been reported to promote wound healing in animals. However, how this process is mediated remains unclear. In this study we examined the mechanisms which underlie the improved wound healing effects of CAP and the roles of associated reactive oxygen and nitrogen species (RONS), which are generated by plasma. By using in vitro models which mimicked various steps of angiogenesis, we demonstrated that CAP triggered the production of nitric oxide (NO), and enhanced cell migration and the assembly of endothelial cells into vessel-like structures. These are both hallmarks of the proliferative phase of wound healing. Using a mouse model of a third-degree burn wound, we went on to show that CAP treatment was associated with enhanced angiogenesis, characterised by accelerated in vivo wound healing and increased cellular proliferation. Here, CAP significantly increased the in vivo production of endothelial NO synthase (eNOS), an enzyme that catalyses NO synthesis in endothelial cells, and significantly increased the expression of pro-angiogenic PDGFRß and CD31 markers in mouse wounds. Mechanistically, we showed that CAP induced eNOS phosphorylation and activation, thereby increasing the levels of endogenous NO in endothelial cells. Increased NO generation facilitated by CAP further stimulated important pro-angiogenic VEGFA/VEGFR2 signalling in vitro. This proof-of-concept study may guide future efforts aimed at addressing the use of physical plasma and its therapeutic applications in a variety of pathological scenarios. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Evaluation of the effectiveness of kINPen Med plasma jet and bioactive agent therapy in a rat model of wound healing.

Chronic nonhealing wounds, particularly those complicated by multidrug resistant infections, represent a major health and economic challenge. Plasma treatment promotes wound repair due to its antimicrobial, angiogenic, and cell modulating properties. This study investigated the efficacy of the kINPen Med system in promoting healing and assessed if efficacy was enhanced by adding collagen or hyaluronic acid (HA). Two 6 mm diameter punch biopsy wounds were created on the lumbar spine of Sprague Dawley rats. Based on the results of a pilot study, operating process conditions involving 30 s plasma/day were selected for the pivotal study. In the pivotal study, six groups of rats (n = 28/group) received either control (1), plasma (2), HA (3), plasma and HA (4), collagen (5), or plasma and collagen (6). Wound measurements were obtained on Days 0, 4, 7, and 14. The mean reduction in wound size was significantly higher in all treatment groups compared to controls on Day 4; group 6 performed best. On Day 7, group 6 still performed significantly better compared to groups 1, 2, 3, and 4. Day 14 results were more comparable between groups. Histology (Day 14) revealed epidermal hyperplasia and serocellular crusts. Neutrophilic infiltrates in group 6 were significantly lower compared to group 2. Mononuclear infiltrates were highest in groups 3 and 5, while Langerhans cells were observed in all groups. These results underpin the clinical benefits of the kINPen Med plasma system, particularly when combined with collagen during early inflammatory phases, and support the conduct of future human clinical trials.

Non­thermal plasma inhibits tumor growth and proliferation and enhances the sensitivity to radiation in vitro and in vivo.

Cancer is a major disease currently endangering the entire world population. Morbidity and mortality have increased substantially during recent decades. Radiotherapy is a primary treatment for malignant tumors, however side­effects and tumor cell resistance to ionizing radiation reduce the efficacy of radiotherapy. In recent years, non­thermal plasma (NTP) technology been used to treat cancer. In this study, we investigated the toxic effects of NTP on normal cells and tumor cells. We explored the inhibitory effect of NTP on tumor cell proliferation and evaluated the radiation­sensitizing effects of NTP on tumor cells and its mechanisms. In short, we examined the effect of NTP­combined radiation on proliferation, the cell cycle, apoptosis and DNA damage in normal and cancer cells. We found that NTP inhibited proliferation and induced apoptosis in tumor cells. NTP was more lethal to tumor cells than to normal cells. We found promising synergies of NTP with radiotherapy on cancer cells owing to their combined cytotoxic effects by generating ROS, inducing cell cycle arrest and apoptosis. NTP may be a new candidate for the treatment of cancer.

Medical applications of cold atmospheric plasma: state of the science.

Cold atmospheric plasmas (CAP) have been used in multiple medical fields and have become a promising medical technology. CAP-generating devices are safe and easy to operate and can now be manufactured at a low cost due to advancements in electronics and microchips. A primary application of CAP is as a broad-spectrum antimicrobial technology. With the high incidence of infections caused by drug-resistant microorganisms, a non-antibiotic based treatment modality such as CAP holds great therapeutic promise, particularly in the wound care field. In addition to its antimicrobial properties, CAP treatment enhances wound healing by increasing cutaneous microcirculation, monocyte stimulation, and keratinocyte proliferation. CAP has been used by dentists for disinfection of teeth, enhancing gingival fibroblast activity, and even teeth whitening. CAP can combat tumour growth by increasing the efficacy of antitumour therapeutic agents, reactivating apoptotic pathways, or down-regulating growth-related gene sites. Most of the health-care related research on CAP has occurred in the past 15 years; the field is relatively young and needs additional research, as well as confirmation of the existing supporting literature. The purpose of this report is to provide the reader with an overview of the therapeutic application of the cold plasma technology.