Cold atmospheric helium plasma in the Post-COVID-19 era: A promising tool for the disinfection of silicone endotracheal prostheses

ABSTRACT Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses.

Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Helium Cold Atmospheric Plasma Causes Morphological and Biochemical Alterations in Candida albicans Cells.

(1) Background: Previous studies reported the promising inhibitory effect of cold atmospheric plasma (CAP) on Candida albicans. However, the exact mechanisms of CAP's action on the fungal cell are still poorly understood. This study aims to elucidate the CAP effect on C. albicans cell wall, by evaluating the alterations on its structure and biochemical composition; (2) Methods: C. albicans cells treated with Helium-CAP were analyzed by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) in order to detect morphological, topographic and biochemical changes in the fungal cell wall. Cells treated with caspofungin were also analyzed for comparative purposes; (3) Results: Expressive morphological and topographic changes, such as increased roughness and shape modification, were observed in the cells after CAP exposure. The alterations detected were similar to those observed after the treatment with caspofungin. The main biochemical changes occurred in polysaccharides content, and an overall decrease in glucans and an increase in chitin synthesis were detected; (4) Conclusions: Helium-CAP caused morphological and topographic alterations in C. albicans cells and affected the cell wall polysaccharide content.

Study of a Conical Plasma Jet with a Cloth-Covered Nozzle for Polymer Treatment.

Although atmospheric pressure plasma jets (APPJs) have been widely employed for materials modification, they have some drawbacks, such as the small treatment area (couple of cm2). To overcome this limitation, a funnel-like APPJ with a wide exit has been proposed. In this work, a gas-permeable cotton cloth covered the nozzle of the device to improve the gas flow dynamics and increase its range of operation. The funnel jet was flushed with Ar, and the plasma was ignited in a wide range of gas flow rates and the gap distances between the exit nozzle and the sample holder. The device characterization included electric measurements and optical emission spectroscopy (OES). To evaluate the size of the treatment and the degree of surface modification, large samples of high-density polyethylene (PE) were exposed to plasma for 5 min. Afterward, the samples were analyzed via water contact angle WCA measurements, scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). It was found that surface modification occurs simultaneously on the top and bottom faces of the samples. However, the treatment incorporated different functional groups on each side.

Non-Thermal Atmospheric Pressure Plasma Application in Endodontics.

The failure of endodontic treatment is frequently associated with the presence of remaining microorganisms, mainly due to the difficulty of eliminating the biofilm and the limitation of conventional irrigation solutions. Non-thermal atmospheric pressure plasma (NTPP) has been suggested for many applications in the medical field and can be applied directly to biological surfaces or indirectly through activated liquids. This literature review aims to evaluate the potential of NTPP application in Endodontics. A search in the databases Lilacs, Pubmed, and Ebsco was performed. Seventeen manuscripts published between 2007 and 2022 that followed our established inclusion criteria were found. The selected manuscripts evaluated the use of NTPP regarding its antimicrobial activity, in the direct exposure and indirect method, i.e., plasma-activated liquid. Of these, 15 used direct exposure. Different parameters, such as working gas and distance from the apparatus to the substrate, were evaluated in vitro and ex vivo. NTPP showed a disinfection property against important endodontic microorganisms, mainly Enterococcus faecalis and Candida albicans. The antimicrobial potential was dependent on plasma exposure time, with the highest antimicrobial effects over eight minutes of exposure. Interestingly, the association of NTPP and conventional antimicrobial solutions, in general, was shown to be more effective than both treatments separately. This association showed antimicrobial results with a short plasma exposure time, what could be interesting in clinical practice. However, considering the lack of standardization of the direct exposure parameters and few studies about plasma-activated liquids, more studies in the area for endodontic purposes are still required.

Simultaneous Treatment of Both Sides of the Polymer with a Conical-Shaped Atmospheric Pressure Plasma Jet.

A conical-shaped atmospheric pressure plasma jet (CS-APPJ) was developed to overcome a standard limitation of APPJs, which is their small treatment area. The CS-APPJs increase the treatment area but use the same gas flow. In the present work, polypropylene samples were treated by CS-APPJ and characterized by scanning electron microscope (SEM), the contact angle, Fourier-transformed infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). It was observed that the treatment co-occurs on the face directly in contact with the plasma and on the opposite face (OF) of the samples, i.e., no contact. However, the treatment changed the chemical composition on each side; the OF is rougher than the direct contact face (DCF), probably due to the oxygen groups in excess at the DCF and nitrogen in quantity at the OF. Although simultaneous treatment of both sides of the sample occurs for most atmospheric plasma treatments, this phenomenon is not explored in the literature.

Different Radial Modification Profiles Observed on APPJ-Treated Polypropylene Surfaces according to the Distance between Plasma Outlet and Target.

The plasma jet transfer technique relies on a conductive wire at floating potential, which, upon entering in contact with a primary discharge, is capable of igniting a small plasma plume at the distal end of a long flexible plastic tube. In this work, two different long tube configurations were employed for the surface modification of polypropylene (PP) samples using argon as the working gas. One of the jet configurations has a thin copper (Cu) wire, which was installed inside the long tube. In the other configuration, the floating electrode is a metallic mesh placed between two plastic tubes in a coaxial arrangement. In the first case, the tip of the Cu wire is in direct contact with the working gas at the plasma outlet, whereas, in the second, the inner plastic tube provides an additional dielectric barrier that prevents the conductor from being in contact with the gas. Water contact angle (WCA) measurements on treated PP samples revealed that different surface modification radial profiles are formed when the distance (d) between the plasma outlet and target is changed. Moreover, it was found that the highest WCA reduction does not always occur at the point where the plasma impinges the surface of the material, especially when the d value is small. Through X-ray photoelectron spectroscopy (XPS) analysis, it was confirmed that the WCA values are directly linked to the oxygen-functional groups formed on the PP surfaces after the plasma treatment. An analysis of the WCA measurements along the surface, as well as their temporal evolution, together with the XPS data, suggest that, when the treatment is performed at small d values, the plasma jet removes some functional groups at the point where the plasma hits the surface, thus leading to peculiar WCA profiles.

Effect of Cold Atmospheric Plasma Jet Associated to Polyene Antifungals on Candida albicans Biofilms.

The increasing incidence of antifungal resistance represents a great challenge in the medical area and, for this reason, new therapeutic alternatives for the treatment of fungal infections are urgently required. Cold atmospheric plasma (CAP) has been proposed as a promising alternative technique for the treatment of superficial candidiasis, with inhibitory effect both in vitro and in vivo. However, little is known on the association of CAP with conventional antifungals. The aim of this study was to evaluate the effects of the association between CAP and conventional polyene antifungals on Candida albicans biofilms. C. albicans SC 5314 and a clinical isolate were used to grow 24 or 48 h biofilms, under standardized conditions. After that, the biofilms were exposed to nystatin, amphotericin B and CAP, separately or in combination. Different concentrations of the antifungals and sequences of treatment were evaluated to establish the most effective protocol. Biofilms viability after the treatments was compared to negative control. Data were compared by One-way ANOVA and post hoc Tukey (5%). The results demonstrate that 5 min exposure to CAP showed more effective antifungal effect on biofilms when compared to nystatin and amphotericin B. Additionally, it was detected that CAP showed similar (but smaller in magnitude) effects when applied in association with nystatin and amphotericin B at 40 µg/mL and 60 µg/mL. Therefore, it can be concluded that the application of CAP alone was more effective against C. albicans biofilms than in combination with conventional polyene antifungal agents.

Cold Atmospheric Plasma Jet as a Possible Adjuvant Therapy for Periodontal Disease.

Due to the limitations of traditional periodontal therapies, and reported cold atmospheric plasma anti-inflammatory/antimicrobial activities, plasma could be an adjuvant therapy to periodontitis. Porphyromonas gingivalis was grown in blood agar. Standardized suspensions were plated on blood agar and plasma-treated for planktonic growth. For biofilm, dual-species Streptococcus gordonii + P. gingivalis biofilm grew for 48 h and then was plasma-treated. XTT assay and CFU counting were performed. Cytotoxicity was accessed immediately or after 24 h. Plasma was applied for 1, 3, 5 or 7 min. In vivo: Thirty C57BI/6 mice were subject to experimental periodontitis for 11 days. Immediately after ligature removal, animals were plasma-treated for 5 min once-Group P1 (n = 10); twice (Day 11 and 13)-Group P2 (n = 10); or not treated-Group S (n = 10). Mice were euthanized on day 15. Histological and microtomography analyses were performed. Significance level was 5%. Halo diameter increased proportionally to time of exposure contrary to CFU/mL counting. Mean/SD of fibroblasts viability did not vary among the groups. Plasma was able to inhibit P. gingivalis in planktonic culture and biofilm in a cell-safe manner. Moreover, plasma treatment in vivo, for 5 min, tends to improve periodontal tissue recovery, proportionally to the number of plasma applications.

Cold Atmospheric Pressure Plasma Jet Reduces Trichophyton rubrum Adherence and Infection Capacity.

This study aimed to evaluate the effects of cold atmospheric pressure plasma (CAPP) jet on Trichophyton rubrum growth, germination and adherence to nail. The effects of plasma jet on T. rubrum conidia germination and on mycelial growth were evaluated by in vitro assays. An ex vivo nail infection model was used to evaluate the effects on conidia adherence and infection. Biochemical analyses of nail fragments exposed or not to CAPP were performed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Plasma jet exposure for 10 and 15 min completely inhibited mycelial growth after only one exposure. Fifteen minutes of exposure could reduce conidia germination in suspension. Fungal suspensions exposed to plasma jet for 10 and 15 min were not able to infect nail specimens. These results were corroborated by ATR-FTIR analyses of nail fragments. In conclusion, single exposure to CAPP for 15 min was able to inhibit fungal growth, adherence and infection capacity. The results suggest that cold atmospheric plasma jet can be a promising alternative for the treatment of onychomycoses caused by T. rubrum.

Amplitude-modulated cold atmospheric pressure plasma jet for treatment of oral candidiasis: In vivo study.

The aim of this study was to establish an effective and safe protocol for in vivo oral candidiasis treatment with atmospheric plasma jets. A novel amplitude-modulated cold atmospheric pressure plasma jet (AM-CAPPJ) device, operating with Helium, was tested. In vitro assays with Candida albicans biofilms and Vero cells were performed in order to determine the effective parameters with low cytotoxicity. After the determination of such parameters, the protocol was evaluated in experimentally induced oral candidiasis in mice. AM-CAPPJ could significantly reduce the viability of C. albicans biofilms after 5 minutes of plasma exposure when compared to the non-exposed group (p = 0.0033). After this period of exposure, high viability of Vero cells was maintained (86.33 ± 10.45%). Also, no late effects on these cells were observed after 24 and 48 hours (83.24±15.23% and 88.96±18.65%, respectively). Histological analyses revealed significantly lower occurrence of inflammatory alterations in the AM-CAPPJ group when compared to non-treated and nystatin-treated groups (p < 0.0001). Although no significant differences among the values of CFU/tongue were observed among the non-treated group and the groups treated with AM-CAPPJ or nystatin (p = 0.3201), histological analyses revealed marked reduction in candidal tissue invasion. In conclusion, these results point out to a clinical applicability of this protocol, due to the simultaneous anti-inflammatory and inhibitory effects of AM-CAPPJ with low cytotoxicity.