Repetitive Bacterial Disinfection of Respirators by Polydopamine Coating

Abstract

To solve the current and future mask shortage problems, developing methods of disinfecting respirators is essential, where none of the existing methods have been successfully utilized until recently. Herein, we introduce a novel method of conferring antibacterial activity to the main filtering material (i.e., polypropylene (PP)) of a respirator through sequential polydopamine (PDA) coatings. Two-step dip-coating in dopamine solution, which corresponds to one complete cycle, produces stable PDA films at the interface of the filtering material, which subsequently locally generates H₂O₂ that can be further transformed into hydroxyl radicals to inactivate pathogens. Specifically, the primary dip-coating creates a scaffold PDA film that acts as a mechanical support, and anchoring dopamine, which substantially produces H₂O₂, is immobilized to the scaffold PDA during the subsequent secondary dip-coating process. The antibacterial activity was confirmed by bacterial tests using Escherichia coli. In short, the number of colonies after incubation of the polypropylene filter with and without the PDA coating in the bacterial solution was compared. The number of bacteria in the PDA-coated sample (0.54 × 10⁹ CFU/mL·cm²) was significantly reduced compared to that in the original PP sample (0.81 × 10⁹ CFU/mL·cm²), demonstrating a positive relationship with the H₂O₂ production. Moreover, this antibacterial ability can be maintained by simply utilizing additional PDA coatings, suggesting that the respirators can be recycled. Finally, the in vitro cytotoxicity was confirmed by the CCK-8 assay, which demonstrated that the PDA-coated PP filter is biocompatible. We believe that the newly proposed method for disinfection of respirators may substitute conventional methods and can be used to alleviate the mask shortage problem.

1. Introduction

Wearing medical-grade masks (e.g., N95) is presumably the easiest and most effective method of preventing the spread of pandemic and epidemic diseases, including the current COVID-19 virus. External pathogens can be efficiently captured in the filtering material (melt-blown polypropylene) of a mask [1]. However, repetitive usage of a single mask may result in unexpected infection from random pathogens, as the inner environment of masks is ideal for pathogen growth. For example, the humidity and temperature of the filtering N95 respirator can increase rapidly to 90% RH (relative humidity) and 30–35 °C after 10 min of usage [2]. The rapid growth of random pathogens may result in pathogenic consequences. However, repetitive usage of medical-grade masks is occasionally inevitable owing to the unexpected shortage of masks [3].

Several methods have been developed to overcome this issue. Thermal disinfection [4,5], ultraviolet germicidal irradiation [6,7], and H₂O₂-based decontamination [8] are representative examples. However, none of these methods have successfully solved the mask shortage problem for the following reasons: (1) The polymeric filtering material in masks undergoes structural deformation during the continuous thermal decontamination process; thus, only a few cycles of decontamination are possible in the thermal disinfection method [9,10]. (2) In the case of ultraviolet germicidal irradiation, the installation of a UV source is essential, and the effectiveness of decontamination varies greatly depending on the distance between the mask and the UV source [11]. (3) Decontamination using vaporized H₂O₂ may be the most reasonable method for disinfection; however, a large amount of H₂O₂ may also result in structural deformation of the filtering materials in masks [9]. It may be ideal to locally produce a small amount of H₂O₂ in masks for disinfection purposes (removal of pathogens). For this purpose, H₂O₂-generating ability should be conferred to the filtering material, melt-blown polypropylene. However, methods of adding new functions (functionalization) to polypropylene are extremely limited owing to the inherent hydrophobicity of polypropylene [12,13].

Polydopamine, inspired by the adhesion mechanism of marine mussels, is a thin multifunctional coating material that can be introduced into any kind of substrate [14,15,16,17,18,19], including polypropylene [20]. The inherent catechol functionality of polydopamine allows various chemical conjugations, such as hydrogen bonding, π-π stacking, metal coordination, and other covalent bonds [21], and is the origin of material-independent coating ability. Moreover, the catechol group has a high potential for transformation into H₂O₂ [22,23], which then generates hydroxyl radicals (OH·) [24] via a chemical oxidation process. Reactive oxygen species (ROS) oxidize the crucial components of pathogens, resulting in disinfection [25]. Thus, we postulate that successive polydopamine coatings may be a way of permanently producing H₂O₂. This is beneficial for avoiding the spontaneous growth of pathogens in the mask, enabling the reuse of masks without structural deformation of the filtering materials.

Herein, we present a method for repetitively imparting antibacterial properties to the conventional N95 mask through polydopamine (PDA) coating. For this purpose, melt-blown polypropylene, which can capture external pathogens when used as a mask filter, is coated with PDA to introduce a catechol functional group. Catechol acts as a raw material for generating H₂O₂, and the generated H₂O₂ exhibits an antibacterial effect owing to the continuous conversion to the hydroxyl radical (OH·). Successful coating is confirmed through X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and other techniques. The antibacterial effect of PP coated with PDA is demonstrated through antibacterial property tests using E. coli (Escherichia coli) and hydrogen peroxide generation. The recyclability of the mask is also demonstrated by confirming that the antibacterial activity and hydrogen peroxide generation are maintained when the PP filter functionalized with PDA is recoated with PDA several times. We expect that this study will enable the development of a semi-permanent mask by imparting antibacterial properties while alleviating the current problem of mask shortage, which is expected to extend into the future.

2. Materials and Methods

2.1. Materials

N95 respirators were purchased from 3M (N95 1860, Saint Paul, MN, USA). Dopamine hydrochloride (99%) and the Pierce Quantitative Peroxide Assay Kit with sorbitol were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hydrochloric (HCl) acid (35–37%) was purchased from Samchun Chemical (Seoul, South Korea). Trizma base and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Burlington, MA, USA). The humidifier was purchased from Opolar (MH304, Shenzhen, China). H₂O₂ (30% stock solution) was purchased from Junsei Chemical Co. (Tokyo, Japan). Phosphate-buffered saline (PBS; autoclaved, pH 7.4) was purchased from Bioneer (South Korea). Escherichia coli was purchased from KCTC (KCTC 52,645, Seoul, South Korea). LB broth miller and LB agar miller were purchased from BD Difco (Franklin Lakes, NJ, USA). Mouse fibroblast cells (L929) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell Counting Kit-8 Assay (CCK-8) was purchased from Dojindo (Kumamoto, Japan). The syringe filter was purchased from Sartorius AG (Göttingen, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM; high glucose) was purchased from Cytiva (Marlborough, MA, USA). The 96-well cell culture plate was purchased from Corning (Corning, NY, USA).

2.2. Polydopamine Coating Method

The PP filters were cut to dimensions (20 × 35 mm²) that fit into a Petri dish (60 × 15 mm²). The four edges of the PP filters were fixed using adhesive tape. To coat the PP filters using the two sequential dip-coating methods, a 20 mL aliquot of dopamine solution (8 mg/mL in 10 mM Tris–HCl buffer, pH 8.5) was added to the Petri dish. The samples were incubated in an orbital shaker (50 rpm) for 24 h at room temperature (RT). The dopamine solution was removed and replaced with a newly prepared dopamine solution (0.8 mg mL⁻¹ dopamine HCl in 10 mM Tris–HCl buffer, pH 8.5) and incubated for an additional 24 h with constant shaking (50 rpm) at RT. The PDA-coated PP filters (PP-PDA1) were washed thrice with acidified water (pH 3.5) to remove unreacted free dopamine. Subsequently, the filters were sterilized with 70% ethanol and air-dried before use.

2.3. Antibacterial Property Tests

The four edges of autoclaved PP-PDA1 and PP without coating (control) placed on a Petri dish were fixed with a thermal resistance masking tape. Escherichia coli (E. coli) grown on LB broth was diluted with sterile PBS to the desired concentration (approximately 3–8 × 10⁹ colony-forming units (CFU)/mL). The bacterial solution (20 mL) was added to each Petri dish. The dishes were then placed inside a chamber, pre-sterilized with 70% ethanol. One hundred microliters of bacterial solution on the PP substrate were collected and used as a control sample. Another 100 μL aliquot of bacterial solution on PP-PDA1, after 9 h of incubation in a chamber at constant temperature and humidity (35 °C, 90%), was also collected (the solution was collected after pipetting more than 20 times to homogeneously disperse the precipitated bacteria in the solution). Both solutions were immediately spread onto agar plates and further incubated for 18–24 h. The number of colonies on the agar plates was manually counted.

2.4. Quantification of the Generated H₂O₂

The H₂O₂ concentration was quantified using a quantitative peroxide assay kit (i.e., ferrous oxidation–xylenol orange assay) according to the guidelines provided by Thermo Fisher Scientific (USA). The absorption intensity of the mixture (100 μL) at 595 nm was measured using a microplate reader (Infinite M Flex, Tecan, Switzerland). A standard curve was prepared using a series of solutions containing 0–0.1 mM of H₂O₂ (0, 11, 17, 33, and 100 μM). The absorption intensity at an identical wavelength (595 nm) was used to generate a standard curve.

2.5. Cell Cytotoxicity Test

Mouse fibroblast cells (L929, American Type Culture Collection, Manassas, VA, USA) and the cell counting kit-8 assay (CCK-8, Dojindo, Kumamoto, Japan) were used for the cell viability tests. PP-PDA1 extract was prepared according to ISO 10993-5 and ISO 10993-12. In short, PP-PDA1 was submerged in DMEM at 37 °C for 9 h, which matched the general mask-wearing time. The extract was filtered using a 0.22 μm pore-sized PTFE syringe filter (Sartorius AG, Göttingen, Germany). A cell suspension of the desired concentration (1.0 × 10⁵ cell/mL) was prepared using DMEM. The cell suspension (100 μL) was seeded into a 96-well cell culture plate and incubated at 37 °C in a 5% CO₂ incubator for 24 h. The medium was then replaced with PP-PDA1 extract and further incubated under 5% CO₂ at 37 °C for another 24 h. After incubation, the cell viability was evaluated using the cell counting kit-8 assay (CCK-8) and a microplate reader (absorbance wavelength of 450 nm). A blank medium was used as a negative control and DMSO was used as a positive control.

2.6. Recycling Tests

PP-PDA1 used in the previous tests was re-coated with PDA using the same two sequential dip-coating methods to produce PP-PDA2 and PP-PDA3 (the numbers indicate the coating cycles). The bacterial colonies were also counted by following the previous description (refer to Section 2.3). H₂O₂ generation in the solution containing PP-PDA2 and PP-PDA3 was also measured by referring to previously described methods (refer to Section 2.4).

2.7. Statistical Analysis

The bacterial and H₂O₂ production experiments were performed 15 or 5 times on the individual samples, and the results are reported as the mean ± standard deviation (SD). The p-values were calculated by unpaired two-tailed t-tests with equal variances using Excel software. The statistical significance was set at p < 0.05.

3. Results and Discussion

The inner condition of the mask (30–35 °C, RH 90%) [2] is an ideal environment for the growth of deadly pathogens; thus, long-term usage of masks results in unexpected risks from pathogens (e.g., bacteria). Figure 1 shows a schematic of the developed method of conferring antibacterial properties to conventional N95 masks via polydopamine (PDA) coating. Two sequential dip-coatings in dopamine hydrochloride solution (8 and 0.8 mg/mL, respectively) developed thick and stable PDA layers on the main filtering part (melt-blown polypropylene), allowing a large amount of H₂O₂ production while breathing [26]. The detailed principle of the antibacterial effects is as follows: when the catechol in the PDA-coated polypropylene filter interacts with oxygen from a breath, catechol can be instantly oxidized and transformed into quinone; reactive oxygen species (ROS), including H₂O₂, are simultaneously produced as by-products [22,23]. The produced H₂O₂ can be released from the human body as non-toxic water. In contrast, the unmanaged H₂O₂ in bacteria can be further transformed into hydroxyl radicals (OH·) [24], resulting in rupture of the lipid membrane in bacteria [25].

XPS was used to demonstrate the successful introduction of PDA into the PP mesh filter. The PDA-coated PP (PP-PDA1, the number indicates the coating cycle) prepared using the two sequential coatings, illustrated in Figure 1, was used for the analysis. Four pieces of evidence of successful PDA coating were observed as follows: (1) A strong N1s peak appeared in the XPS profile of PP-PDA1, whereas a nitrogen peak was observed in that of bare PP (Figure 2a,b). (2) The atomic ratio of the C1s peak decreased significantly (from 98.2% to 75.7%) after PDA coating, which should be the consequence of screening of bare PP. (3) The atomic ratio of C1s, N1s, and O1s in the PP-PDA1 sample (10:1:2) matched well with the theoretical atomic ratio of PDA (8:1:2). (4) From the high-resolution C1s peak, bare PP was composed of 98.7% C-C and 1.3% C–O, whereas the composition of the oxygenated/nitrogenated functional groups notably increased to 30.1% (-C-O/C-N), 16.0% (-C=O), and 15.9% (-COOH) (Figure 2c,d and Table S1). As a result of the successful PDA coating, a clear color change was observed at the surface. The black color is a distinctive signal of melanin-like PDA owing to its inherent molecular structure that absorbs various ranges of visible light [27]. SEM was used to visually identify the polydopamine-coated layer of PP-PDA1. The polydopamine layer itself has a thickness of tens of nanometers and is difficult to distinguish using SEM. However, the presence of polydopamine can be indirectly verified through the adhesion of the polydopamine nanoparticles spontaneously generated during polydopamine synthesis [28]. Nanoparticles were observed in the polydopamine-coated PP filter (Figure 2f). These results confirmed that PP was successfully coated with PDA.

Mimicking the practical conditions in masks is essential for achieving reliable antibacterial activity of the PDA-coated PP filter. According to a previous report [2], we performed all relevant experiments, including bacterial inoculation and H₂O₂ production experiments, in a specialized chamber, maintaining a humidity of 90% at a constant temperature of 35 °C (Figure 3a). For the bacterial growth experiment, PP-PDA1 was dipped into E. coli solution with a concentration of 3–8 × 10⁹ colony-forming units (CFU)/mL. The concentration of E. coli was calculated after 9 h of incubation (see Supplementary Materials for the detailed procedures). The bacterial level decreased significantly in PP-PDA1 (0.54 × 10⁹ ± 0.08 CFU/mL·cm², n = 5) compared to the initial bacterial concentration in the control sample (bare PP, 0.81 × 10⁹ ± 0.09 CFU/mL·cm², n = 15).

To demonstrate the correlation between H₂O₂ generation and the antimicrobial properties of the mask, the amount of H₂O₂ in each sample was quantified. For this, the samples were submerged in PBS solution with a pH of 7.4, with gentle shaking (50 rpm), as described in a previous study [26], and the ferrous oxidation–xylenol orange (FOX) assay was utilized to quantify the exact amount of generated H₂O₂ (see the Supplementary Materials for the detailed procedures). It was confirmed that the PP-PDA1 produced 18.11 ± 0.74 μM/cm² (n = 5) of H₂O₂ after 9 h of incubation. This suggests that the antibacterial properties of PP-PDA1 originate from H₂O₂ production.

An in vitro cell viability test was conducted using the CCK-8 assay to demonstrate that the PDA-coated mask does not have a toxic effect. A blank medium was used as a negative control (NC), and DMSO was used as a positive control (PC). The relative cell viability was calculated based on the NC. The cell viability results from the PC (14.02% ± 5.88%, n = 5) demonstrated that the test results were reliable. According to the ISO 10993-5 method which we performed, a relative cell viability of >80% is defined as the condition with no cytotoxicity [29]. The relative cell viability in PP-PDA1 was 84.11% ± 11.34% (n = 5), indicating that the cytotoxicity of PP-PDA1 was low. According to the results, it is believed that PDA-coated PP should be a non-cytotoxic and safe material for use in masks.

H₂O₂ production in PDA is not permanent and has a saturation point [26]. The advantage of the developed system is that the H₂O₂ source (catechols) can be easily refilled by additional PDA dip-coating cycles. Thus, the antibacterial activity of the mask can be maintained by continuous PDA coatings, which are virtually unlimited, as illustrated in Figure 4a. To test this hypothesis, additional bacterial growth experiments were performed by utilizing PDA-coated PP meshes with 1, 2, and 3 coating cycles, and the corresponding samples are designated as PP-PDA1, PP-PDA2, and PP-PDA3, respectively (Figure 4b). The bacterial concentration in each substrate after 9 h of incubation was similarly diminished from 0.81 × 10⁹ ± 0.09 CFU/mL·cm² (control, n = 15) to 0.54 × 10⁹ ± 0.08 CFU/mL·cm² (n = 5, PP-PDA1), 0.69 × 10⁹ ± 0.17 CFU/mL·cm² (n = 5, PP-PDA2), and 0.52 × 10⁹ ± 0.12 CFU/mL·cm² (n = 5, PP-PDA3), respectively. Thus, the antibacterial ability can be constantly sustained in every single substrate regardless of the coating cycles, demonstrating antibacterial recyclability.

To demonstrate that the antibacterial ability is proportionally correlated with H₂O₂ production, we quantified the amount of H₂O₂ in each sample with different coating cycles. In the PP sample subjected to a single PDA coating cycle (PP-PDA1), 18.11 ± 0.74 μM/cm² (n = 5) of H₂O₂ was generated in the PBS solution after 9 h of incubation. The amount of H₂O₂ was comparable in the other samples (PP-PDA2 and PP-PDA3), with values of 21.29 ± 1.39 μM/cm² (n = 5) and 16.69 ± 4.17 μM/cm² (n = 5) (Figure 4c). The above results suggest that the antibacterial property of PDA-coated PP can be sustained by the constant introduction of the catechol functionality through additional PDA coatings.

4. Conclusions

We introduced an accessible method of conferring antibacterial properties to respirators via polydopamine (PDA) coatings, which locally generate H₂O₂ from the filtering material polypropylene (PP). The ability of catechol to provide H₂O₂ is easily restored by another PDA coating cycle. As a result, the PDA-coated PP samples show comparable antibacterial ability regardless of the number of coating cycles, suggesting that respirators can be permanently reused. In this regard, our method is expected to be preferred over the conventional thermal disinfection method, which easily causes physical deformation of the PP mesh within a limited cycle. The in vitro cytotoxicity of the PDA-coated PP was also confirmed through the CCK-8 assay: the cell viability was more than 80%, which indicates that the materials are biocompatible. Theoretically, other catecholic dip-coatings (e.g., poly(tannic acid), poly(catechin)) sharing a similar molecular structure to polydopamine are expected to provide antibacterial properties. Ideally, teas or wines with abundant catecholic building blocks that can self-assemble into polyphenolic macromolecules, such as dopamine, may be used to produce DIY versions of recyclable masks. The proposed dip-coating method is more accessible and affordable than other conventional methods that typically require special equipment (e.g., a UV light source for sterilization irradiation). Taken together, our method can prospectively substitute the conventional methods of bacterial disinfection and provide a breakthrough for alleviating the ongoing and future mask shortage associated with the pandemic.