Evaluation of supercritical CO2 sterilization efficacy for sanitizing personal protective equipment from the coronavirus SARS-CoV-2

Abstract

The purpose of this research is to evaluate the supercritical carbon dioxide (scCO₂) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a "worst-case" regarding challenged PPE. The inactivation of the microbes by scCO₂ sterilization with NovaKill or H₂O₂ sterilant was investigated as a function of exposure times ranging from 5 to 90 min with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO₂-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO₂ sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.

Keywords: Bacillus atrophaeus endospore; COVID-19; Coronavirus; Face mask; HCoV-NL63; Supercritical carbon dioxide.

Graphical abstract

Fig. 1

Schematic diagram of the mask reprocessing experiment in the pipeline: include sample preparation, inoculation setup, NovaClean process in the NovaSterilis equipment (have CO₂ cylinder, syringe pump, pump header cooler, and heater, preheating coil, heater, extraction vessel, and water bath) and characterization and analysis of sanitization.

Fig. 2

Extracellular factors analysis of B. atrophaeus spores upon NovaClean process. Spore inactivation by scCO₂-based NovaClean (NovaKill) process. Fluorescence micrographs of spore (A), and germinated spore (B) stained with Live/Dead (BacLight staining reagent) for membrane integrity assessment. The spores were incubated for 20 min in the presence of mixtures of SYTO™ 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide. Viable spores were stained green, and spores with compromised membranes were stained red. The scale bar is 5 μM for all images, and the micrographs in all pictures are at the same scale. (C) SEM pictures of untreated (i) and NovaClean processed (ii) spores. (D) EDX of respective B. atrophaeus spores before and after NovaClean process. EDX line profile was digitally magnified for clear visualization. (E) DPA release of spores treated by scCO₂, NovaKill, and H₂O₂. (mean ± SD, n = 3). The additive effects were compared with scCO₂ treatment sample using ordinary one-way ANOVA with Tukey's multiple comparisons test in GraphPad Prism 9. *Significantly different from scCO₂ treatment. * p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Inactivation of human coronavirus using the NovaClean process. The data were expressed as the mean ± standard deviation (S.D.). Each viral load was compared using an unpaired t-test. *Significantly different from control. **** p < 0.0001. ND = not detected.

Fig. 4

3D surface mapping of N95 (A), 3-layer surgical mask (B), and cloth mask (C). A-1, B-1, and C-1, surface damage assessment for A, B, and C mask respectively by using gel-based photometric profilometry. Scale bars = 500 μm. A-2, B-2, and C-2, analysis of fiber distribution (provided with height and distance in micrometer) for A, B, and C mask respectively by using ImageJ. With the ImageJ software, a representative fiber image shows the longitudinal line drawn across from the mask surface non-flat region that defines the region of interest (ROI) for the pre-and-post NovaClean process. The gray pixel intensities in ROI were quantified for fiber distribution in the individual mask surface and illustrated histograms.

Fig. 5

Water contact angle measurement of three different masks (A) before reprocessing and (B) after reprocessing cycles. The mask contact angle was compared to each other using ordinary one-way ANOVA with Tukey's multiple comparisons test. *Significantly different from others. ** p < 0.001.

Fig. 6

The sequence of morphological and functional changes occurring at different stages of NovaClean process. Sanitization by scCO₂ based NovaClean process induces CO₂ and radical elements penetration in cells and disrupts cellular integrity. Continuous exposure to NovaClean process causes the germination and functional defect via intracellular component leaks, pH-dependent disintegration of the membrane protein and damage membrane integrity. After 40 min, all cells are completely inactivated. Scale bars, spore and virus fine arts representations = 200 nm.