Page 464 - IJB-10-5

International Journal of Bioprinting 3D-printed plasma devices for decontamination

In this study, we examined the efficiency of three CAP 3). However, H O likely played a significant role in our
2
2
devices for selecting enveloped and nonenveloped viruses analysis of bacterial inactivation in saline solution because
and a clinically important gram-negative bacterium. The of the low solubility of RONS in water. In contrast to the
three devices were produced via a 3D printer and could be long CAP exposure times required for virus inactivation
easily adjusted to the desired size, shape, and inner chamber (up to 120 min; Figures 7 and 8), bacterial cells were
geometry. Along with the original device used in earlier inactivated by devices 2 and 3 within 1 min (Figure 5),
studies, we tested two optimized versions containing a where evaporation of the liquid is not expected to be a
closed chamber that produces a more densely concentrated relevant factor. The antibacterial effect of CAP was also
particle environment. These versions incorporated a reflected by a greater degree of physical damage, including
tubular “volcano” design, which accurately directs particle membrane impairment and cell content leakage, as
flow toward the sample. Another feature of potential visualized via TEM. This damage was more rapidly inflicted
improvement was the use of two electric discharges in by devices 2 and 3 (Figure 6). CAP exposure damages the
parallel (Figure 1). All three devices demonstrated good bacterial cell wall, leading to high levels of oxidative stress
performance in decontaminating surfaces from pathogens, and cell membrane rupture, as well as bacterial cell death
but the devices displayed different efficiencies defined by due to DNA damage. 60–64 Many studies have reported
the time of exposure to CAP required for inactivation. pronounced antibacterial effects of CAP generated in
According to the parameters recorded, optimized devices gaseous environments (i.e., containing a single RONS or a
2 and 3 were superior to the original device 1 in all analyses combination of RONS), which produces H O and NO - in
performed, including the reduction in infectivity, nucleic aqueous environments. 58,65–69 2 2 2
acid degradation, and external membrane damage in
relation to the duration of CAP exposure. The antiviral efficiency exhibited a similar trend as
described above, with devices 2 and 3 outperforming
To characterize the CAP composition produced by device 1 in all the assays performed (Figures 7 and 8;
individual devices, we analyzed the concentration of Tables S1–S4). Given the long exposure time required for
RONS accumulated in gaseous (Figures 2 and 3) and viral inactivation (up to 120 min), significant evaporation
aqueous (Figure 4) environments. The optimized devices of the liquid surrounding the viral particles is likely.
yielded relatively high concentrations of O , N O, and Hence, both RONS detected in a gaseous environment
3
2
NO in the gaseous environment and relatively high and H O detected at high concentrations in the aqueous
2
2
2
H2O2 concentrations in the saline solution. The pH of environment upon treatment by devices 2 and 3 appear to
the exposed saline drop was generally low, with the lowest play a relevant role in the virucidal effects observed. Viral
value generated by device 3 (pH 0.5). All the tested devices infectivity decreased gradually over the CAP exposure
-
-
yielded comparable concentrations of NO and NO in time, and complete elimination was generally achieved
3
2
the aqueous environment (based on SEM images). The more rapidly with the optimized devices. The reduction in
results indicate that the use of closed-chamber formats viral genome copy number, which was analyzed to monitor
(devices 2 and 3) led to changes in the CAP atmosphere, the physical integrity of virus particles, exhibited the
thereby increasing decontamination efficiency. Notably, same trend, albeit more slowly, indicating that infectivity
the inclusion of an additional discharge electrode (device was diminished before complete particle disintegration.
2) had a minimal impact on decontamination efficiency. This trend was previously reported by us and supports
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The antibacterial properties of CAP apparently the notion that perfect virus fitness viability is required
correlate with the concentration of RONS produced by for infectivity but does not directly correlate with particle
the devices. This notion is reflected by the greater efficacy integrity. Repeated measurements often reveal fluctuating
of optimized devices 2 and 3 in eliminating the viability readouts, especially for HAdV and HRV, at shorter CAP
of P. aeruginosa almost instantaneously, whereas device exposure times. This was also the case for the control
1 required 30 min for the same effect (Figure 5). In this measurements before CAP exposure, thus reflecting the
study, bacteria suspended in saline solution during CAP biological complexity of the experimental system. In line
treatment suggested that RONS, specifically H2O2, may with previous observations, enveloped RNA viruses
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play a crucial role in antibacterial efficacy, especially in (SARS-CoV-2 and IAV in the present study) and the
direct bacterial exposure to CAP. This assumption is nonenveloped virus HRV were more rapidly inactivated
56
supported by the considerably greater H O concentration by CAP, whereas HAdV displayed a greater degree
2
2
in the CAP-exposed saline drop generated by devices 2 of stability. It has been previously reported that both
and 3 (Figure 4). The antimicrobial effect of RONS is well enveloped and nonenveloped viruses can be inactivated
described in the literature, 57–59 and their presence has also by reactive plasma particles through oxidative damage to
been validated in a gaseous environment (Figures 2 and proteins, viral envelopes (if present), and nucleic acids.
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Volume 10 Issue 5 (2024) 456 doi: 10.36922/ijb.3679